CRITICAL CARE AND TRAUMA
SECTION EDITOR
PETER M. SUTER
Ketamine Suppresses Proinflammatory Cytokine
Production in Human Whole Blood In Vitro
Takashi Kawasaki, MD, Masanori Ogata, MD, Chika Kawasaki,
Yoshitaka Inoue, MD, and Akio Shigematsu, MD
Department of Anesthesiology, University of Occupational and Environmental Health, Kitakyushu, Japan
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The production of proinflammatory cytokines, such as
tumor necrosis factor (TNF) a, interleukin (IL)-6, and
IL-8, increases in patients with sepsis; marked produc-
tion causes organ failure and septic shock. We previ-
ously reported that ketamine suppressed lipopolysac-
charide (LPS)-induced TNF-a production in mice.
However, there are no reports on the effect of ketamine
on cytokine production in human whole blood. There-
fore, in this study, we investigated the efficacy of ket-
amine on LPS-induced TNF-a, IL-6, and IL-8 produc-
tion and recombinant human (rh) TNF-a–induced IL-6
and IL-8 production in human whole blood. After add-
ing different doses of ketamine to whole blood, the
blood was stimulated with LPS or rhTNF. After incuba-
tion, the plasma TNF-a activity and IL-6 and IL-8 con-
centrations were measured using the L929 cell cytotoxic
C
ytokines are essential for hematopoiesis and im-
mune responses, and they play a key role in the
defense against infection. Lipopolysaccharide
(LPS) is a potent inducer of the inflammation involved in
the pathogenesis of septic shock. It has been demon-
strated that proinflammatory cytokines, such as tumor
necrosis factor (TNF) a, interleukin (IL)-6, and IL-8, in-
crease in patients with sepsis, trauma, and burns (1–6).
IL-6 mediates the acute-phase response, and IL-8 is a
potent chemotactic agent for neutrophils. These cyto-
kines are associated with the development of septic
shock and organ dysfunction. Ketamine, an IV anes-
thetic, has been advocated for anesthesia in septic or
severely ill patients because of its cardiovascular-
stimulating effects (7,8). We previously reported that
ketamine suppressed LPS-induced TNF-a production
This work was supported in part by Grant-in-Aid for Scientific
Research 10671453 from the Ministry of Education, Science, Sport
and Culture of Japan.
Presented in part at the annual meeting of the American Society
of Anesthesiologists, October 17–21, 1998, Orlando, FL.
Accepted for publication May 10, 1999.
Address correspondence and reprint requests to Masanori Ogata,
MD, Department of Anesthesiology, University of Occupational and
Environmental Health, 1–1-1 Iseigaoka Yahata-nishi-ku Kitakyushu,
807-8555, Japan. Address e-mail to mogata@med.uoeh-u.ac.jp.
©1999 by the International Anesthesia Research Society
0003-2999/99
MD, Jun-ichi Ogata, MD,
assay or an enzyme-linked immunoassay. Ketamine
significantly suppressed LPS-induced TNF-a produc-
tion at concentrations .20 mg/mL. At concentrations
.100 mg/mL, ketamine also significantly suppressed
both LPS-induced and rhTNF-induced IL-6 and IL-8
production. In this study, we demonstrated that ket-
amine directly inhibits the production of proinflamma-
tory cytokines such as TNF-a, IL-6, and IL-8 in human
whole blood. Implications: We found that ketamine
suppressed lipopolysaccharide-induced tumor necro-
sis factor a, interleukin (IL)-6, and IL-8 production and
recombinant human tumor necrosis factor-induced
IL-6 and IL-8 production in human whole blood. Ket-
amine directly suppresses proinflammatory cytokine
production.
(Anesth Analg 1999;89:665–9)
and mortality in carrageenan-sensitized endotoxin shock
mice (9,10). However, there are no reports on the effects
of ketamine on the production of other proinflammatory
cytokines. Not only does LPS trigger the production of
TNF-a, IL-6, and IL-8, it also stimulates IL-6 and IL-8
production (11).
Therefore, in this study, we investigated the effects
of ketamine in human whole blood on LPS-induced
production of TNF-a, IL-6, and IL-8 and on TNF-a–
induced IL-6 and IL-8 production.
Methods
Phenol-extracted Escherichia coli (serotype 0127: B8)
LPS was purchased from Difco Laboratories (Detroit,
MI). Ketamine (10 mg/mL) was purchased from
Sankyo Pharmaceutical Co. (Tokyo, Japan). Recombi-
nant human (rh) TNF-a was purchased from Gen-
zyme Co. (Cambridge, MA).
After approval of our human investigations commit-
tee, informed consent was obtained from 15 healthy
male volunteers not taking any medication. Blood sam-
ples were drawn into tubes containing heparin and di-
luted with 5 vol of RPMI 1640 (Nissui Pharmaceutical,
Anesth Analg 1999;89:665–9 665
666 CRITICAL CARE AND TRAUMA KAWASAKI ET AL.
KETAMINE SUPPRESSES TNF-a, IL-6, AND IL-8 PRODUCTION IN HUMAN BLOOD
Tokyo, Japan) (12). One milliliter of diluted blood per
well was placed into 24-well tissue culture plates.
After different doses (0 –500 mg/mL) of ketamine
were added to each well, whole blood was stimulated
with LPS (10 ng/mL). The blood was then incubated
for 6 or 12 h at 37°C in a 95% air/5% CO2 incubator.
After incubation, the blood was centrifuged at 700g for
10 min to remove blood cells. Supernatant samples
were collected and stored at 280°C until assayed.
To assess the direct effect of ketamine on IL-6 and
IL-8 production, we studied the effect of ketamine on
rhTNF-induced IL-6 and IL-8 production. After differ-
ent doses (0 –500 mg/mL) of ketamine were added to
each well, whole blood was stimulated with rhTNF-a
(104 U/mL). The dose of rhTNF-a chosen induced the
same level of TNF-a activity as when 10 ng/mL LPS
was used on L929 cells in the preliminary study. The
blood was then incubated for 6 h at 37°C in a 95%
air/5% CO2 incubator. After incubation, the blood was
centrifuged at 700g for 10 min to remove blood cells.
The supernatant was collected and stored at 280°C
until assayed.
The L929 cell cytotoxic assay (described previously)
was used to determine the plasma TNF-a activity (13).
Briefly, L929 cells in RPMI 1640 medium containing
5% fetal calf serum were seeded at 3 3 105 cells/well
in 96-well flat-bottomed microtiter plates (Becton
Dickinson, Lincoln Park, NJ) and incubated overnight
at 37°C in an atmosphere of 5% CO2 in air. Serial
1:2 dilutions of samples were made in this me-
dium containing 1 mg/mL actinomycin D (Banyu
Pharmaceutical Co., Tokyo, Japan), and 0.1 mL of each
dilution was added to different wells. On the follow-
ing day, cell survival was assessed by fixing and stain-
ing the cells with crystal violet (0.2% in 20% metha-
nol), and 1% sodium dodecyl sulfate was added to
each well to solubilize the stained cells. The absor-
bance of each well was determined at 490 nm using a
microplate reader (Bio-Rad Laboratories, Richmond,
CA). TNF activity was expressed in units per milliliter,
which is the reciprocal of the dilution necessary for
50% lysis of the cells.
The plasma IL-6 concentration was measured in
duplicate using a commercially available enzyme-
linked immunoassay (IL-6 Enzyme Immunoassay Kit;
Advanced Magnetics, Inc., Cambridge, MA). The
intra- and interassay precision was 9% and 6%, respec-
tively, at an IL-6 concentration of 88 pg/mL. The
plasma IL-8 concentration was measured in duplicate
using the same enzyme-linked immunoassay. The
intra- and interassay precision was 7% and 4%, respec-
tively, at an IL-8 concentration of 76 pg/mL. Accord-
ing to the manufacturer, cross-reactivity with other
cytokines is negligible in both assays.
To assess the effect of ketamine on leukocyte viabil-
ity, different doses (0 –500 mg/mL) of ketamine were
added to diluted human whole blood and incubated
ANESTH ANALG
1999;89:665–9
for 5 h at 37°C in a 95% air/5% CO2 incubator. After
incubation, blood was centrifuged at 700g for 10 min.
Buffy coats were isolated and NH4Cl lysis of red blood
cells was performed. The white blood cells were re-
suspended in RPMI 1640 medium containing 5% fetal
calf serum, and the cells were stained with 0.2%
trypan blue. The cell survival rate was assessed by
microscope.
All data are presented as the mean 6 sem. The
repeated paired t-test was used for statistical analysis
to compare values with the control value. A significant
difference was presumed at a probability value of
,0.05.
Results
Whole blood was stimulated using different doses of
LPS (0 –100 ng/mL). LPS induced TNF-a production
in human whole blood in a dose-dependent manner at
concentrations of 0.1–10 ng/mL. TNF-a production
reached a plateau with LPS doses $10 ng/mL (Fig-
ure 1). Therefore, we used an LPS concentration of 10
ng/mL in our experiments.
After adding different doses (0–500 mg/mL) of ket-
amine, whole blood was stimulated with LPS (10 ng/
mL) (n 5 15). Figure 2A shows the effect of ketamine on
LPS-induced TNF-a production. When the blood was
incubated for 6 h, ketamine significantly suppressed
LPS-induced TNF-a production in a dose-dependent
manner at concentrations of 20–500 mg/mL, compared
with the control (P , 0.05). Concentrations ,4 mg/mL
ketamine had no effect on LPS-induced TNF-a produc-
tion. When the blood was incubated for 12 h, LPS-
induced TNF-a production increased from 1152 6 176
U/mL (6-h incubation) to 2176 6 174 U/mL. At concen-
trations of 20–500 mg/mL, ketamine significantly sup-
pressed LPS-induced TNF-a production (P , 0.05).
Figure 2B shows the effect of ketamine on LPS-
induced IL-6 production. Ketamine (100 –500 mg/mL)
suppressed LPS-induced IL-6 production in the 6-h
incubation (P , 0.0001). Ketamine (100 –500 mg/mL)
also suppressed LPS-induced IL-8 production com-
pared with the control (P , 0.05) (Figure 2C). Concen-
trations ,20 mg/mL ketamine had no effect on IL-6 or
IL-8 production in the 6-h whole blood incubation
(Figure 2, B and C). When the blood was incubated for
12 h, LPS-induced IL-6 production increased from
555 6 33 pg/mL (6-h incubation) to 1195 6 59 pg/mL,
and IL-8 production increased from 907 6 56 pg/mL
(6-h incubation) to 1502 6 165 pg/mL. Ketamine (100 –
500 mg/mL) also significantly suppressed LPS-
induced IL-6 and IL-8 production when the blood was
incubated for 12 h (P , 0.0001) (Figure 2, B and C).
After adding different doses (0 –500 mg/mL) of ket-
amine, whole blood was stimulated with rhTNF
(104 U/mL) (n 5 8). Figure 3 shows the cytokine
ANESTH ANALG CRITICAL CARE AND TRAUMA KAWASAKI ET AL. 667
1999;89:665–9 KETAMINE SUPPRESSES TNF-a, IL-6, AND IL-8 PRODUCTION IN HUMAN BLOOD

Figure 1. Lipopolysaccharide (LPS)-induced tumor necrosis factor

(TNF) a production in human whole blood. Values are expressed as
mean 6 sem (n 5 5).

production when the whole blood was incubated with

rhTNF for 6 h. Ketamine 250 and 500 mg/mL signifi-
cantly suppressed rhTNF-induced IL-6 production
(P , 0.05) (Figure 3A), whereas ,100 mg/mL ket-
amine had no effect on rhTNF-induced IL-6 produc-
tion (Figure 3A). Ketamine (100 –500 mg/mL) also sig-
nificantly suppressed rhTNF-induced IL-8 production
(P , 0.0001) (Figure 3B), whereas ,20 mg/mL ket-
amine had no effect on rhTNF-induced IL-8 produc-
tion (Figure 3B).
Ketamine had no effect on L929 cell viability. Ket-
amine also had no effect on white blood cell viability, as
assessed by the exclusion of the vital stain trypan blue.

Discussion
In this study, we demonstrated that ketamine sup-
pressed both LPS-induced TNF-a, IL-6, and IL-8 produc-
tion and rhTNF-induced IL-6 and IL-8 production in
human whole blood. TNF-a is the first cytokine ex-
pressed after LPS stimulation, after which it stimulates
IL-6 and IL-8 secretion from macrophages, monocytes,
neutrophils, and endothelial cells. We suspected that the
suppressive effect of ketamine on LPS-induced IL-6 and
IL-8 production might be caused by the inhibiting effect
of ketamine on LPS-induced TNF-a production. How-
ever, our results demonstrated that ketamine suppressed
rhTNF-induced IL-6 and IL-8 production, which sug-
gests that the suppression of IL-6 and IL-8 production is
dependent not only on the suppression of TNF-a, but
also on the direct effect of ketamine on the production of
these cytokines in whole blood.
The mechanism for the suppressive effect of ket-
amine on cytokine production is not clear. A detect-
able increase in the TNF-a mRNA level is seen min-
utes after the LPS injection and reaches a peak two
hours after the LPS injection (11). In a previous study,
we reported that the addition of ketamine two hours
Figure 2. Effect of ketamine on lipopolysaccharide (LPS)-induced tu-
mor necrosis factor (TNF) a (A), interleukin-6 (B), and interleukin-8 (C)
production. After ketamine (0–500 mg/mL) was added, human whole
blood was stimulated by LPS (10 ng/mL) and incubated for 6 or 12 h.
Values are expressed as mean 6 sem (n 5 15). *P , 0.05, #P , 0.0001
compared with control.
after the LPS stimulation effectively suppressed TNF
production (9). We assume that ketamine may regu-
late LPS-induced TNF-a production at a posttran-
scriptional level. Further study is required to elucidate
the mechanism for the suppressive effect of ketamine
on TNF-a, IL-6, and IL-8.
In this study, human whole blood was used as an ex
vivo model of cytokine production in human tissue. Us-
ing the whole blood model reduces the confounding
668 CRITICAL CARE AND TRAUMA KAWASAKI ET AL.
KETAMINE SUPPRESSES TNF-a, IL-6, AND IL-8 PRODUCTION IN HUMAN BLOOD
Figure 3. Effect of ketamine on recombinant human tumor necrosis
factor (rhTNF) a-induced interleukin-6 (A) and interleukin-8 (B)
production. After ketamine (0 –500 mg/mL) was added, human
whole blood was stimulated by rhTNF-a and incubated for 6 h.
Values are expressed as mean 6 sem (n 5 8). *P , 0.05, #P , 0.0001
compared with control.
factors that may be associated with the isolation of
monocytes, such as the adherence-induced expression of
cell-surface TNF or TNF mRNA (14). Moreover, whole
blood is a more physiologic environment in which to
examine cytokine production in response to LPS because
the cellular interactions are preserved and the presence
of LPS-binding protein is maintained (15).
In our study, after a 12-hour incubation, LPS-induced
TNF-a, IL-6, and IL-8 production in human whole blood
increased significantly compared with a 6-hour incuba-
tion. In contrast, Deforge et al. (11) demonstrated that
IL-6 reached a plateau six hours after the LPS challenge.
They also showed that IL-8 increased after LPS stimula-
tion, reached a plateau between 6 and 12 hours, then
continued to increase for .24 hours (16). The time course
for LPS-induced cytokine production that we observed
differed from theirs. In this study, we stimulated human
whole blood at an LPS concentration of 10 ng/mL,
whereas Deforge et al. used an LPS concentration of
10 mg/mL. The response to a low concentration of LPS
has been attributed to the binding of an LPS-LPS binding
ANESTH ANALG
1999;89:665–9
protein complex to CD14 on the monocyte surface,
whereas the response to LPS concentrations .10 ng/mL
can occur in the absence of either LPS binding protein or
CD14 (15). We assume that different cytokine responses
may be caused by the differences in the LPS concen-
tration.
It has been demonstrated that ketamine, an IV an-
esthetic, has protective effects in septic patients and
in an animal septic shock model (7–10,17,18). The
cardiovascular-stimulating effects of ketamine have
been advocated for anesthesia in septic patients (7,8).
The concentration of ketamine in human plasma
reached 110 mM by the IV administration of ketamine
2–2.2 mg/kg (19). Shimaoka et al. (20) showed that
ketamine (30 – 600 mM) inhibits nitric oxide production
in mouse-activated macrophage-like cells via inhibi-
tion of TNF-a production, and Li et al. (21) showed
that .10 mM ketamine also inhibits nitric oxide pro-
duction in LPS-treated rat alveolar macrophages.
Schmidt et al. (22) showed that ketamine (10 mg/kg
body weight) also inhibits endotoxin-induced leuko-
cyte adherence due to the decreased production of
TNF-a in rat mesenteric venules. We previously dem-
onstrated that ketamine has a potent suppressive ef-
fect on LPS-induced TNF-a production in vitro and in
vivo (9,10).
In addition, in the present study, we showed that
.20 mg/mL ketamine (73 mM) suppressed LPS-
induced TNF-a production and that .100 mg/mL ket-
amine (365 mM) had a potent suppressive effect on
IL-6 and IL-8 production in human whole blood.
These studies suggest that the protective effects of
ketamine in septic patients and in animal septic shock
models are due to suppression of the excessive pro-
duction of proinflammatory cytokines.
Roytblat et al. (23) reported that a single dose of
ketamine 0.25 mg/kg administered before cardiopul-
monary bypass suppressed the increase in serum IL-
6 during and after coronary artery bypass surgery,
and that a subanesthetic dose of ketamine suppressed
IL-6 production in women undergoing hysterectomy
(24). However, in our study, such a small dose of
ketamine did not suppress LPS- and rhTNF-induced
IL-6 production. The reason for this is not clear but
may lie in the differences between in vivo and in vitro
experimental conditions.
In conclusion, we demonstrated that ketamine di-
rectly inhibits the production of proinflammatory cy-
tokines such as TNF-a, IL-6, and IL-8 in human whole
blood. Further study is required to elucidate the mech-
anism of the suppressive effect of ketamine.
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