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© 1999 International Anesthesia Research Society
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Abstract |
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The production of proinflammatory cytokines, such as tumor necrosis factor (TNF)
, interleukin (IL)-6, and IL-8, increases in patients with sepsis; marked production causes organ failure and septic shock. We previously reported that ketamine suppressed lipopolysaccharide (LPS)-induced TNF- production in mice. However, there are no reports on the effect of ketamine on cytokine production in human whole blood. Therefore, in this study, we investigated the efficacy of ketamine on LPS-induced TNF-, IL-6, and IL-8 production and recombinant human (rh) TNF-–induced IL-6 and IL-8 production in human whole blood. After adding different doses of ketamine to whole blood, the blood was stimulated with LPS or rhTNF. After incubation, the plasma TNF- activity and IL-6 and IL-8 concentrations were measured using the L929 cell cytotoxic assay or an enzyme-linked immunoassay. Ketamine significantly suppressed LPS-induced TNF- production at concentrations >20 µg/mL. At concentrations >100 µg/mL, ketamine also significantly suppressed both LPS-induced and rhTNF-induced IL-6 and IL-8 production. In this study, we demonstrated that ketamine directly inhibits the production of proinflammatory cytokines such as TNF-, IL-6, and IL-8 in human whole blood.
Implications: We found that ketamine suppressed lipopolysaccharide-induced tumor necrosis factor , interleukin (IL)-6, and IL-8 production and recombinant human tumor necrosis factor-induced IL-6 and IL-8 production in human whole blood. Ketamine directly suppresses proinflammatory cytokine production.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Cytokines are essential for hematopoiesis and immune 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 demonstrated that proinflammatory cytokines, such as tumor necrosis factor (TNF)
, interleukin (IL)-6, and IL-8, increase 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 cytokines are associated with the development of septic shock and organ dysfunction. Ketamine, an IV anesthetic, 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- production 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-, 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-, IL-6, and IL-8 and on TNF-–induced IL-6 and IL-8 production.
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Methods |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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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). Recombinant human (rh) TNF-
was purchased from Genzyme Co. (Cambridge, MA). After approval of our human investigations committee, informed consent was obtained from 15 healthy male volunteers not taking any medication. Blood samples were drawn into tubes containing heparin and diluted with 5 vol of RPMI 1640 (Nissui Pharmaceutical, Tokyo, Japan) (12). One milliliter of diluted blood per well was placed into 24-well tissue culture plates.
After different doses (0–500 µg/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 -80°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 different doses (0–500 µg/mL) of ketamine were added to each well, whole blood was stimulated with rhTNF- (104 U/mL). The dose of rhTNF- chosen induced the same level of TNF- 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 -80°C until assayed.
The L929 cell cytotoxic assay (described previously) was used to determine the plasma TNF- activity (13). Briefly, L929 cells in RPMI 1640 medium containing 5% fetal calf serum were seeded at 3 x 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 medium 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 following day, cell survival was assessed by fixing and staining the cells with crystal violet (0.2% in 20% methanol), and 1% sodium dodecyl sulfate was added to each well to solubilize the stained cells. The absorbance 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%, respectively, 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%, respectively, at an IL-8 concentration of 76 pg/mL. According to the manufacturer, cross-reactivity with other cytokines is negligible in both assays.
To assess the effect of ketamine on leukocyte viability, different doses (0–500 µg/mL) of ketamine were added to diluted human whole blood and incubated 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 resuspended 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 ± 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.
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Results |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Whole blood was stimulated using different doses of LPS (0–100 ng/mL). LPS induced TNF-
production in human whole blood in a dose-dependent manner at concentrations of 0.1–10 ng/mL. TNF- production reached a plateau with LPS doses 
10 ng/mL (Figure 1). Therefore, we used an LPS concentration of 10 ng/mL in our experiments.
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After adding different doses (0–500 µg/mL) of ketamine, whole blood was stimulated with LPS (10 ng/mL) (n = 15). Figure 2A shows the effect of ketamine on LPS-induced TNF-
production. When the blood was incubated for 6 h, ketamine significantly suppressed LPS-induced TNF- production in a dose-dependent manner at concentrations of 20–500 µg/mL, compared with the control (P < 0.05). Concentrations <4 µg/mL ketamine had no effect on LPS-induced TNF- production. When the blood was incubated for 12 h, LPS-induced TNF- production increased from 1152 ± 176 U/mL (6-h incubation) to 2176 ± 174 U/mL. At concentrations of 20–500 µg/mL, ketamine significantly suppressed LPS-induced TNF- production (P < 0.05).
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Figure 2B shows the effect of ketamine on LPS-induced IL-6 production. Ketamine (100–500 µg/mL) suppressed LPS-induced IL-6 production in the 6-h incubation (P < 0.0001). Ketamine (100–500 µg/mL) also suppressed LPS-induced IL-8 production compared with the control (P < 0.05) (Figure 2C). Concentrations <20 µg/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 ± 33 pg/mL (6-h incubation) to 1195 ± 59 pg/mL, and IL-8 production increased from 907 ± 56 pg/mL (6-h incubation) to 1502 ± 165 pg/mL. Ketamine (100–500 µg/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 µg/mL) of ketamine, whole blood was stimulated with rhTNF (104 U/mL) (n = 8). Figure 3 shows the cytokine production when the whole blood was incubated with rhTNF for 6 h. Ketamine 250 and 500 µg/mL significantly suppressed rhTNF-induced IL-6 production (P < 0.05) (Figure 3A), whereas <100 µg/mL ketamine had no effect on rhTNF-induced IL-6 production (Figure 3A). Ketamine (100–500 µg/mL) also significantly suppressed rhTNF-induced IL-8 production (P < 0.0001) (Figure 3B), whereas <20 µg/mL ketamine had no effect on rhTNF-induced IL-8 production (Figure 3B).
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Ketamine had no effect on L929 cell viability. Ketamine also had no effect on white blood cell viability, as assessed by the exclusion of the vital stain trypan blue.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In this study, we demonstrated that ketamine suppressed both LPS-induced TNF-
, IL-6, and IL-8 production and rhTNF-induced IL-6 and IL-8 production in human whole blood. TNF- is the first cytokine expressed 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- production. However, our results demonstrated that ketamine suppressed rhTNF-induced IL-6 and IL-8 production, which suggests that the suppression of IL-6 and IL-8 production is dependent not only on the suppression of TNF-, but also on the direct effect of ketamine on the production of these cytokines in whole blood.
The mechanism for the suppressive effect of ketamine on cytokine production is not clear. A detectable increase in the TNF- mRNA level is seen minutes 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 after the LPS stimulation effectively suppressed TNF production (9). We assume that ketamine may regulate LPS-induced TNF- production at a posttranscriptional level. Further study is required to elucidate the mechanism for the suppressive effect of ketamine on TNF-, IL-6, and IL-8.
In this study, human whole blood was used as an ex vivo model of cytokine production in human tissue. Using the whole blood model reduces the confounding 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-, IL-6, and IL-8 production in human whole blood increased significantly compared with a 6-hour incubation. 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 stimulation, 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 µg/mL. The response to a low concentration of LPS has been attributed to the binding of an LPS-LPS binding 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 concentration.
It has been demonstrated that ketamine, an IV anesthetic, 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 µM by the IV administration of ketamine 2–2.2 mg/kg (19). Shimaoka et al. (20) showed that ketamine (30–600 µM) inhibits nitric oxide production in mouse-activated macrophage-like cells via inhibition of TNF- production, and Li et al. (21) showed that >10 µM ketamine also inhibits nitric oxide production in LPS-treated rat alveolar macrophages. Schmidt et al. (22) showed that ketamine (10 mg/kg body weight) also inhibits endotoxin-induced leukocyte adherence due to the decreased production of TNF- in rat mesenteric venules. We previously demonstrated that ketamine has a potent suppressive effect on LPS-induced TNF- production in vitro and in vivo (9,10).
In addition, in the present study, we showed that >20 µg/mL ketamine (73 µM) suppressed LPS-induced TNF- production and that >100 µg/mL ketamine (365 µM) 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 production of proinflammatory cytokines.
Roytblat et al. (23) reported that a single dose of ketamine 0.25 mg/kg administered before cardiopulmonary 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 directly inhibits the production of proinflammatory cytokines such as TNF-, IL-6, and IL-8 in human whole blood. Further study is required to elucidate the mechanism of the suppressive effect of ketamine.
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Acknowledgments |
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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.
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Footnotes |
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Presented in part at the annual meeting of the American Society of Anesthesiologists, October 17–21, 1998, Orlando, FL.
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References |
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