J Infect Chemother (2010) 16:94–99 DOI 10.1007/s10156-009-0012-5
ORIGINAL ARTICLE
Effect of linezolid on cytokine production capacity and plasma endotoxin levels in response to lipopolysaccharide stimulation of whole blood Gaku Takahashi • Nobuhiro Sato • Yasunori Yaegashi • Masahiro Kojika Naoya Matsumoto • Tomohiro Kikkawa • Tatsuyori Shozushima • Shinji Akitomi • Kiichi Aoki • Naoko Ito • Koichi Hoshikawa • Yasushi Suzuki • Yoshihiro Inoue • Go Wakabayashi • Shigeatsu Endo
•
Received: 11 May 2009 / Accepted: 19 September 2009 / Published online: 22 January 2010 Ó Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases 2010
Abstract The purpose of this study was to assess lipopolysaccharide (LPS)-stimulated cytokine production in the presence of linezolid (LZD) in comparison with the drug effect on the plasma endotoxin level. Peripheral venous whole-blood samples collected from five healthy subjects were stimulated with 10 lg/ml of LPS. LZD was then added to the LPS-stimulated blood samples at concentrations of 0, 2, 4, and 15 lg/ml, followed by incubation for 24 h at 37°C in a 5% CO2–95% air atmosphere. Supernatants of the resultant cultures were assayed to determine the levels of tumor necrosis factor (TNF)-a, interferon (IFN)-c, interleukin (IL)-10, monocyte chemoattractant protein (MCP)-1, and endotoxin. Significant decreases in the levels of TNF-a and IFN-c were observed in the LZD 2, 4, and 15 lg/ml groups as compared with that in the 0 lg/ml group (Dunnett’s procedure; P \ 0.05). The level of IL-10 tended to increase irrespective of the LZD concentration; however, no significant intergroup differences were observed [analysis of variance (ANOVA); P = 0.68]. No significant decrease of the endotoxin level was observed in the LZD 2, 4, or 15 lg/ml groups as
G. Takahashi N. Sato Y. Yaegashi M. Kojika (&) N. Matsumoto T. Kikkawa T. Shozushima S. Akitomi K. Aoki N. Ito K. Hoshikawa Y. Suzuki Y. Inoue S. Endo Department of Critical Care Medicine, Iwate Medical University, School of Medicine, 19-1 Uchimaru, Morioka 020-8505, Japan e-mail:
[email protected] G. Wakabayashi Department of Surgery, Iwate Medical University, School of Medicine, Morioka, Japan
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compared with that in the 0 lg/ml group, with no significant intergroup differences (ANOVA; P = 0.83). No change in the MCP-1 levels was observed irrespective of the LZD concentration (ANOVA; P = 0.82). To conclude: (1) it appears possible that LZD inhibits the production of INF-c and TNF-a to a limited extent; (2) LZD did not exert any inhibitory effect on endotoxin production by bacteria, while suppressing cytokine production. The results indicate that LZD may have a significant role in saving the lives of patients with sepsis. Keywords Cytokine Endotoxin Lipopolysaccharide Sepsis Linezolid
Introduction Linezolid (LZD) is a synthetic oxazolidinone antimicrobial agent with potent activity against gram-positive bacteria, exerted via the inhibition of bacterial protein synthesis [1, 2]. It is well distributed in tissues following administration, and its use is recommended in the treatment of pneumonia caused by methicillin-resistant Staphylococcus aureus (MRSA) [3]. The usefulness of LZD in clinical settings has been demonstrated [4, 5]. In an in vitro study, LZD has been documented to inhibit the production of cytokines from peripheral blood monocytes (PBMCs) of healthy subjects induced by bacterial lipopolysaccharide (LPS) [6]. Antibacterial agents are initially administered empirically in the early stage of treatment of sepsis, prior to identification of the causative pathogen. In other words, empiric therapy is undertaken for the control of simple gram-positive, gram-negative, or mixed gram-positive/ gram-negative bacterial infections. Treatment is then
J Infect Chemother (2010) 16:94–99
switched to the appropriate antibacterial agents as soon as the causative microorganism is identified, or after a few days of clinical monitoring. Because LZD has been shown, although only in vitro to date, to inhibit cytokine production induced by LPS, it was speculated that, in addition to its antibacterial activity, LZD may also exert an inhibitory effect on the cytokine production induced by LPS, irrespective of whether the etiologic agent is gram-positive or gram-negative. The purpose of this study was to compare and assess cytokine production and plasma endotoxin levels in the presence of LZD in a cytokine-producing milieu produced by gram-negative bacterial infection, which was reproduced in vitro by stimulation with LPS.
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Statistical analysis Differences were analyzed by analysis of variance (ANOVA). When a significant difference was found by ANOVA, differences among groups were checked by Dunnett’s procedure (control group, 0 lg/ml group). P values of \0.05 were considered to indicate statistical significance. All statistical analyses were performed on a personal computer with the statistical package, JMP 6.0.3 for Macintosh (SAS Institute, Cary, NC, USA).
Results TNF-a levels at each LZD concentration in cultures 24 h after stimulation
Materials and methods Sample collection and stimulation Peripheral venous whole-blood samples collected from five healthy subjects were stimulated with LPS (Escherichia coli 0111; Sigma, St Louis, MO, USA) added at the concentration of 10 lg/ml, and LZD was then added to aliquots of the LPS-stimulated blood samples at concentrations of 0, 2, 4, or 15 lg/ml, followed by incubation for 24 h at 37°C in a 5% CO2–95% air atmosphere. The supernatants (3000 rpm for 40 s) of the resultant cultures were stored frozen at -80°C and subjected to cytokine assays. The concentrations of LZD employed were based on the pharmacokinetic data stated in the package insert for this pharmaceutical product, viz., a mean peak plasma level of 15 lg/ml and a mean trough plasma level of 3.68 lg/ml in healthy volunteers dosed with LZD at 600 mg every 12 h.
The TNF-a levels after 24 h of incubation in the presence of graded concentrations of LZD in the supernatants of cultures stimulated with LPS were 13355.1 ± 2224.6 pg/ ml in the control group, 8471.9 ± 4336.7 pg/ml in the 2 lg/ml group, 8114.1 ± 3088.1 pg/ml in the 4 lg/ml group, and 7139.9 ± 2656.1 pg/ml in the 15 lg/ml group (ANOVA; P \ 0.05). There were significant differences between the values in the 4 lg/ml group and 15 lg/ml group and that in the control group (Dunnett’s procedure; P \ 0.05; Fig. 1). pg/ml 18000
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Cytokine assays The levels of tumor necrosis factor (TNF)-a, interferon (IFN)-c, interleukin (IL)-10, and monocyte chemoattractant protein (MCP)-1 were determined using Bio-Plex Cytokine Assay Kits (Bio-Rad Laboratories, Hercules, CA, USA); the minimum detectable concentration with these kits is 1.0 pg/ml. Endotoxin assays Endotoxin levels were determined with a Toxinometer, using Endotoxin-Single Wako (Wako Pure Chemical Industries, Osaka, Japan) [7]. The cutoff level for the diagnosis of endotoxemia was 1.1 pg/ml [8].
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Fig. 1 The tumor necrosis factor-a (TNF-a) levels (mean ± SD) after 24 h of incubation in the presence of graded concentrations of linezolid (LZD) in the supernatants of cultures stimulated with lipopolysaccharide (LPS). The TNF-a levels were significantly higher in the control (0 lg/ml) group (P \ 0.05; ANOVA). There were significant differences between the values in the 4 lg/ml group and 15 lg/ml group and that in the control (0 lg/ml) group (Dunnett’s procedure; *P \ 0.05). The TNF-a levels before culture (without LPS, without LZD) were in the range of not more than 1.0 pg/ml (the minimum detectable dose). Horizontal lines in the boxes show median values
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Fig. 2 The interferon-c (INF-c) levels (mean ± SD) after 24 h of incubation in the presence of graded concentrations of LZD in the supernatants of cultures stimulated with LPS. The IFN-c levels were significantly higher in the control (0 lg/ml) group (P \ 0.01; ANOVA). There were significant differences between the values in the 2, 4, and 15 lg/ml groups and that in the control (0 lg/ml) group (Dunnett’s procedure; **P \ 0.01). The INF-c levels before culture (without LPS, without LZD) were in the range of not more than 1.0 pg/ml (the minimum detectable dose). Horizontal lines in the boxes show median values
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INF-c levels at each LZD concentration in cultures 24 h after stimulation The INF-c levels after 24 h of incubation in the presence of graded concentrations of LZD in the supernatants of cultures stimulated with LPS were 1561.8 ± 606.1 pg/ml in the control group, 768.2 ± 285.7 pg/ml in the 2 lg/ml group, 742.5 ± 278.9 pg/ml in the 4 lg/ml group, and 680.4 ± 178.2 pg/ml in the 15 lg/ml group (ANOVA; P \ 0.01). A significant difference was observed between the control group and each of the 2, 4, and 15 lg/ml groups (Dunnett’s procedure; P \ 0.01; Fig. 2). IL-10 levels and MCP-1 levels at each LZD concentration in cultures 24 h after stimulation The IL-10 levels after 24 h of incubation in the presence of graded concentrations of LZD in the supernatants of cultures stimulated with LPS were 133.3 ± 61.9 pg/ml in the control group, 179.1 ± 63.2 pg/ml in the 2 lg/ml group, 177.5 ± 103.0 pg/ml in the 4 lg/ml group, and 193.3 ± 88.0 pg/ml in the 15 lg/ml group (ANOVA; P = 0.68). Although there were no significant intergroup differences, the IL-10 levels tended to be elevated and not decreased in the 2, 4, and 15 lg/ml groups as compared with the level in the control group (Fig. 3a). The MCP-1 levels after 24 h of incubation in the presence of graded concentrations of LZD in the supernatants of
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LZD dose Fig. 3 Interleukin-10 (IL-10; a) and monocyte chemoattractant protein-1 (MCP-1; b) levels after 24 h of incubation in the presence of graded concentrations of LZD in the supernatants of cultures stimulated with LPS. There were no significant differences between the 0, 2, 4, and 15 lg/ml groups in the IL-10 or MCP-1 levels. The IL-10 levels before culture (without LPS, without LZD) were in the range of not more than 1.0 pg/ml (the minimum detectable dose). The MCP-1 levels before culture (without LPS, without LZD) were 31.5 ± 17.5 pg/ml. Horizontal lines in the columns show median values
cultures stimulated with LPS were 2189.9 ± 952.8 pg/ml in the control group, 2682.5 ± 1197.9 pg/ml in the 2 lg/ml group, 2784.6 ± 1650.9 pg/ml in the 4 lg/ml group, and 3106.6 ± 2071.6 pg/ml in the 15 lg/ml group (ANOVA; P = 0.82; Fig. 3b). Endotoxin levels at each LZD concentration in cultures 24 h after stimulation The endotoxin levels after 24 h of incubation in the presence of graded concentrations of LZD in the supernatants of cultures stimulated with LPS were 1013.8 ± 502.4 pg/ml in the control group, 1193.2 ± 709.9 pg/ml in the 2 lg/ml group, 1020.0 ± 658.3 pg/ml in the 4 lg/ml group, and 1300.0 ± 725.6 pg/ml in the 15 lg/ml group (ANOVA; P = 0.83; Fig. 4).
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Fig. 4 The endotoxin levels after 24 h of incubation in the presence of graded concentrations of LZD in the supernatants of cultures stimulated with LPS. There were no significant differences between the 0, 2, 4, and 15 lg/ml groups in the endotoxin levels. The endotoxin levels before culture (without LPS, without LZD) were in the range of not more than 1.1 pg/ml (the minimum detectable dose). Horizontal lines in the boxes show median values
Discussion Initial antimicrobial drug therapy is of vital importance in the treatment of sepsis, and when concurrent immunodeficiency is also taken into account, appropriate drugs must be administered from the outset [9]. Particularly in cases of severe sepsis or septic shock at emergency intensive care units, antimicrobial agents are initiated as empiric therapy even before any causative microorganism can be identified. That is, empiric therapy is undertaken to combat simple gram-positive, gram-negative, and mixed gram-positive/ gram-negative bacterial infections. The excessive release and activity of endogenous inflammatory mediators often give rise to multiple organ failure in patients with sepsis. Endotoxins are among the important pathogenic substances in patients with gram-negative bacterial infections [10, 11]. If a causal focus of infection can be surgically resectable, its excision and drainage should be performed to control the infective focus. Further, the appropriate timing of antimicrobial drug therapy is also of importance; Kumar et al. [12] have reported that the lifesaving rate diminishes by 7.6% with each hour of delay in initiating the antimicrobial therapy. As for the mechanism of action of LZD, the drug exerts its actions by inhibiting protein synthesis; its target consists of bacterial protein synthesis in the early stage of bacterial infection and the drug specifically binds to the ribosomal 50S subunit, thereby inhibiting initiation of the 70S complex to exert its antibacterial activity [1, 2]. LZD has been demonstrated to inhibit cytokine production when applied in the presence of LPS [6]; thus, the drug seems to exert
cytokine-inhibitory activity in addition to its antibacterial activity against gram-positive bacteria. The aim of the present investigation was to evaluate the relationship between the inhibition of cytokine production by LZD and the plasma endotoxin level. This is considered to be of considerable experimental significance, in that LZD may be of clinical benefit even in the management of patients with gram-negative bacterial infections, by inhibiting cytokine production. The cytokines examined in this study were: INF-c and TNF-a as Th1 cytokines, IL-10 as a Th2 cytokine, and MCP-1 as a chemokine. All of these cytokines are important mediators involved in the pathophysiology of sepsis. The TNF-a level was significantly reduced in the LZD 4 and 15 lg/ml groups as compared to that in the control group. The result was essentially consistent with previous reports [6, 13]. The INF-c level was also significantly decreased in the 2, 4, and 15 lg/ml groups as compared with that in the control group. These data indicate that LZD inhibits the production of the Th1 cytokines. The level of IL-10, which acts as an anti-inflammatory cytokine, did not differ significantly among the various LZD concentration groups, but it tended to be elevated in the 2, 4, and 15 lg/ ml groups as compared with the level in the control group. The results show a favorable characteristic of LZD, in that it lowers the levels of inflammatory cytokines and raises the levels of anti-inflammatory cytokines under the condition of sepsis. The endotoxin level after 24 h of incubation after stimulation with 10 lg/ml of LPS showed no appreciable change irrespective of the LZD concentration, whether control, 2, 4, or 15 lg/ml. The present data indicate that LZD exerts an inhibitory effect on cytokine production, while having no inhibitory effect on the production of endotoxins by the causative bacteria. LPS derived from gram-negative bacteria has been shown to induce INF-c production in dendritic cells and macrophages via TLR4. INF-c stimulates macrophages and vascular endothelial cells to produce IL-1 and TNF-a. Such TLR-mediated reactions are required for activation of the Th1 system [14–16]. In response to stimulation by TNF-a, adhesion molecules such as intercellular adhesion molecule (ICAM)-1 are expressed, which facilitate infiltration of the site of inflammation by activated neutrophils [17]. Furthermore, TNF-a stimulates monocytes/macrophages and vascular endothelial cells to produce MCP-1, resulting in the activation of monocytes/macrophages and the induction of IL-6 and other cytokines [18]. Although the present study was only an in vitro study, whole-blood samples were used so as to induce with LPS a state of cytokine elevation similar to that seen in sepsis caused by gram-negative bacterial pathogens [19, 20]. It is considered possible that the suppression of INF-c and TNF-a observed in such a milieu represents a TLR-mediated,
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selective inhibition/modification of Th1 cytokines, as explained above. It would also be reasonable to infer that the drug may inhibit the phosphorylation of signal transducers and activators of transcription 4 (STAT4), which induces Th1 cytokines in the intracellular signaling process, but may have no effect on STAT6, which induces Th2 cytokines [21]. In the present study, LZD failed to cause inhibition of MCP1, suggesting that the inhibitory effect of LZD on the production of Th1 cytokines possibly represents an effect of the drug at limited sites upstream of the inflammatory process. The present in vitro study yielded only limited results. LZD exerts antimicrobial activity against gram-positive bacteria. However, its inhibitory activity against cytokine production decreases following stimulation with LPS. This implies that empiric therapy with LZD initiated prior to the identification of any causative microorganism in a patient with severe sepsis or septic shock may afford clinical benefit against simple gram-positive, gram-negative, and mixed gram-positive/gram-negative infections. The cytokineinhibiting action of LZD appears to inhibit the organ damage induced by hypercytokinemia in septic states and seems to be beneficial clinically. The finding that LZD exerted no inhibitory effect on endotoxin production indicates the necessity of undertaking appropriate surgical treatment of the primary focus of the gram-negative bacterial infection and initiating treatment with appropriate drugs effective against the causative gram-negative bacteria. In cases of sepsis secondary to mixed infection by gram-positive/gramnegative bacteria, however, the cytokine-suppressive effect of LZD is also considered to be advantageous from the viewpoint of recovery from sepsis. In addition, the finding that LZD exerted a cytokine-inhibiting effect means that it would exhibit a cell-inhibiting effect in patients, and a negative effect on being exerted by LZD administration in immunosuppressed states must be considered. What is important in the use of antimicrobial drugs, not merely of LZD, is to administer a drug or drugs to which the causative organism, if identified, is sensitive, with due caution exercised against the emergence of resistant organisms by the administration of appropriate medication, based on the pharmacokinetics/pharmacodynamics [22] of the drug. Acknowledgments This study was supported by grants from the Mutual Aid Corporation for Private Schools of Japan; the Ministry of Education, Culture, Sports, Science, and Technology of Japan; the Ministry of Health, Labour and Welfare of Japan; and the Marumo Emergency Medical Research Foundation.
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