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NetForce Co., Ltd.Computer Technology Integration Co., Ltd., Aichi, Department of Obstetrics and Gynecology, Hamamatsu University School of Medicine, Shizuoka
Department of Knowledge-Based Information Engineering, Toyohashi University of Technology, Toyohashi, Japan
Background.
Lipopolysaccharide (LPS) is the primary mediator of gram-negative sepsis; it induces the production of macrophage-derived cytokines. It has been shown that bikunin, a Kunitz-type protease inhibitor, inhibits LPS-induced cytokine expression.
Methods.
To explore the role of bikunin, bikunin knockout (Bik-/-) mice were used for in vitro cytokine experiments and in vivo animal models.
Results.
We show that a higher level of LPS-mediated death was induced in Bik-/-, compared with wild-type (wt), mice; the administration of bikunin caused a significant reduction in LPS-induced lethality; LPS significantly increased tumor necrosis factor (TNF) and interleukin-1 levels in Bik-/-, relative to wt, mice after LPS challenge; concomitant administration of bikunin inhibited the LPS-induced plasma levels of these cytokines; bikunin suppressed the LPS-induced up-regulation of cytokine expression through the suppression of the phosphorylation of ERK1/2, JNK, and p38 in macrophages; and LPS-induced up-regulation of TNF- expression was not enhanced in Bik-/- macrophages without endogenous bikunin.
Conclusions.
These data allow us to speculate that the increased sensitivity of Bik-/- mice to LPS-induced death in vivo is due to a lack of circulating bikunin in plasma. Bikunin may play a role as a potent anti-inflammatory agent.
Inter-inhibitor (II) is composed of 2 heavy chains and 1 light chain, which is also known as bikunin [1, 2]. Bikunin carries a chondroitin sulfate chain to which the heavy chains are covalently linked [2]. The majority (>95%) of circulating bikunin in plasma is found in the complex form of II and pre-inhibitor. Free bikunin is found only in small amounts. This glycoprotein is considered to be a subunit of II proteins that is cleaved and subsequently cleared rapidly from the body via the kidney [1, 2]. Furthermore, bikunin is composed of a ligand-binding domain (amino terminus) for cell-associated bikunin-binding sites [3, 4] and a protease inhibitory domain (carboxyl terminus) [5] that effectively inhibits trypsin, plasmin, and granulocyte elastase. In addition to its protease inhibitory effects, bikunin plays a role in inhibiting the MAP-kinase (MAPK) signaling cascade [68], which results in the suppression of the expression of several target molecules, including urokinase-type plasminogen activator, which is responsible for tumor cell invasiveness [9], and several cytokines that are responsible for inflammation. Suppression of signaling activation can account for the prevention by bikunin of cancer and inflammation.
Research from various directions has clarified many aspects of the biological function of proteins of the II family and provides a new view of their interesting structure-function relationships, especially regarding their roles in inflammation [2] and cancer [6, 10]. A growing amount of evidence has demonstrated that II is an anti-inflammatory agent: circulating II levels have been found to be lower in patients with severe sepsis than in healthy volunteers [11, 12]. Furthermore, human II may be a useful predictive marker and potential therapeutic agent in sepsis. However, such mechanisms do not readily explain the anti-inflammatory function of bikunin, which has been largely unaddressed.
The pivotal role of tumor necrosis factor (TNF) in inflammation and the potent anti-inflammatory activity of bikunin raise the question of whether the induction of TNF- during inflammation serves as a target for bikunin. To explore the critical role of endogenous bikunin, we used bikunin knockout (Bik-/-) mice [13]. In the present study, we investigated whether the increased sensitivity of Bik-/- mice to lipopolysaccharide (LPS)induced death in vivo is due to a lack of circulating bikunin in plasma. Our findings provide new insights into the mechanism of protection against inflammatory diseases by bikunin.
MATERIALS AND METHODS
Materials.
Bikunin was purified to homogeneity from human urine. A highly purified preparation of human bikunin and polyclonal antibodies to human bikunin was supplied by Mochida Pharmaceutical. The COOH-terminal fragment of bikunin (HI-8) was purified as described elsewhere [5]. The HI-8 construct possesses antiprotease activity [4, 5].
Treatment of mice.
C57Bl/6 and Bik-/- mice matched for sex (female) and age (1015 weeks) were used in the LPS (Escherichia coli serotype O111:B4; Sigma-Aldrich; 1 mg LPS corresponds to 1,000,000 endotoxin units) shock experiments. Mice were intraperitoneally (ip) injected with bikunin at a dose of 0, 25, or 50 mg/kg or with HI-8 at a dose of 100 mg/kg. The highest plasma level was obtained with 125I-bikunin at 3 h after injection with a 7.4% injected dose/g. This corresponds to 50 g/mL (1.25 mol/L) at 3 h after injection. Under the assumption of a 2-compartment model with a 4-parameter fit, the mean -phase half-life for 125I-bikunin was 0.82 h, and the mean -phase half-life was 6.4 h (data not shown). After 1 h, LPS at a dose of 5, 10, or 30 mg/kg in saline was injected ip into mice, and their survival was monitored. Bik-/- mice were obtained from Mochida Pharmaceutical and were fed ad libitum with regular chow (unless otherwise stated). Animal experiments in the present study were performed in compliance with the guidelines of the Institute for Laboratory Animal Research, Hamamatsu University School of Medicine.
Preparation of macrophages and cell culture.
Elicited peritoneal macrophages were obtained from female mice 4 days after ip inoculation of 1 mL of 10% thioglycollate broth [14]. Cells were seeded at 1.5 × 106 cells/well in 6-cm plates. After incubation for 1 h, nonadherent cells were removed by washing the wells twice with RPMI 1640HEPES, and remaining cells were cultured and stimulated for different periods of time in RPMI 1640 medium that contained 10% fetal calf serum (FCS) or bikunin-depleted FCS. To prepare bikunin-depleted FCS, FCS was incubated with antibikunin antibody and protein Gsepharose at 4°C for 4 h. After a brief centrifugation (at 1000 g for 1 min), supernatant was recovered and used as bikunin-depleted FCS. Cells were counted by use of a hemocytometer, and viability was assessed by trypan blue staining.
Cell activation was performed in RPMI-HEPES that contained serum and LPS in the absence or presence of bikunin at the concentrations indicated above for different time periods at 37°C in 5% CO2. Supernatants were harvested for the measurement of TNF- accumulation.
Generation of mice deficient in the bikunin gene.
Mice deficient in bikunin and their genotyping have been described elsewhere [13, 15]. Bikunin deficiency is not neonatally lethal in mice. Under specific pathogen-free conditions, Bik-/- mice survive at the expected Mendelian ratio, remain healthy, and survive to adulthood [13, 15]. There was no difference in blood cell composition between wild-type (wt) and Bik-/- mice. Necropsy and microscopic examination of major tissues revealed no significant pathology in Bik-/- mice (unpublished data).
Assay of plasma levels of TNF-, interleukin (IL)1, and IL-6.
Mice pretreated with bikunin or not pretreated were injected ip with LPS at a dose of 10 mg/kg. Then, with mice under mild anesthesia with diethyl ether at the indicated times after LPS injection, we took blood from the heart into heparinized syringes. Blood was collected 1 h later for the assay of TNF- and 5 h later for the assay of IL-1 or IL-6. Plasma was separated by centrifugation at 5000 g for 10 min. Concentrations of TNF-, IL-1, and IL-6 in plasma were determined with immunoassay kits (TFB).
Western blot analysis .
For the analysis of ERK1/2, JNK, and p38 MAPK phosphorylation, Western blot was performed as described elsewhere [16, 17].
Statistical analysis.
The nonparametric Mann-Whitney U test was used to determine differences between 2 groups, and statistical significance was accepted at P < .05. Statistical differences in survival curves among the groups of mice were analyzed by log-rank test. The Instat software package (version 3; GraphPad Software) was used for statistical analyses.
RESULTS
Increased susceptibility of Bik-/- mice to LPS-induced death.
In Bik-/- mice, LPS (30 mg/kg) induced 90% lethality by 6 h after challenge (figure 1B), whereas wt mice all survived (figure 1A). Twelve hours after LPS challenge (30 mg/kg), all 10 of 10 mice had died, compared with 7 of 10 wt mice. At 24 h after LPS injection, the survival rate was 0% for Bik-/- mice, compared with 10% for wt mice. At 24 h after treatment with 10 mg/kg LPS, lethality was 20% for wt mice (figure 2A), compared with 90% for Bik-/- mice (figure 2B). Bik-/- mice were found to exhibit significantly higher mortality than wt mice after the ip injection of LPS at a dose of 10 or 30 mg/kg. Even at the dose of 5 mg/kg, the survival rate was significantly lower in Bik-/- mice than in wt mice (data not shown). At all of the LPS concentrations tested, a higher rate of death was induced in Bik-/-, compared with wt, mice.
Bikunin protects against bacterial LPS-induced lethality.
It has previously been shown that bikunin inhibits LPS-induced cytokine expression in vitro [18]. Therefore, we investigated whether bikunin can protect against LPS-induced lethality in wt mice in vivo (figures 1 and 2). The ip injection of 30 mg/kg LPS into wt mice resulted in the death of 90% (9/10) of mice within 24 h after injection (figure 1A). In contrast, 6 and 4 of 10 mice were rescued by the ip administration of 25 and 50 mg/kg, respectively, of bikunin (P = .121 and P = .028, respectively). This significant difference in mortality demonstrates that bikunin protects against LPS-induced death.
We also investigated whether the administration of bikunin would cause a significant reduction in LPS-induced lethality in Bik-/- mice (figures 1B and 2B). The susceptibility of Bik-/- mice was almost comparable to that of wt mice, when Bik-/- mice were pretreated with an ip injection of bikunin (25 or 50 mg/kg). These results suggest a critical role of exogenous bikunin in protecting mice from LPS-induced death.
In a parallel experiment, we tried to determine whether the protease inhibitory activity of bikunin is crucial in the reduction of LPS-induced lethality. For this experiment, we used HI-8, which is an active fragment for protease inhibitors [4, 5] but is not recognized by the cell-associated bikunin-binding sites [4]. In contrast to the results with bikunin, HI-8 did not suppress LPS-induced lethality at concentrations of HI-8 as high as 100 mg/kg (figures 1 and 2), which suggests that the activity of bikunin does not depend on its protease inhibitory function.
To determine the effect of exogenous bikunin on LPS-induced proinflammatory cytokine levels, Bik-/- mice were injected with LPS in the absence or presence of bikunin, and cytokine levels were determined at 1 h after LPS injection for TNF- (figure 3A) and at 5 h after LPS injection for IL-1 (figure 3B) and IL-6 (figure 3C). Bikunin significantly reduced cytokine expression in a dose-dependent manner. The administration of 50 mg/kg bikunin together with 10 mg/kg LPS largely inhibited the LPS-induced maximum levels of TNF-, IL-1, and IL-6, by 65%, 49%, and 74%, respectively. We also showed that exogenous bikunin abrogates the LPS-induced up-regulation of cytokine expression in wt mice (data not shown). The proinflammatory cytokines TNF- and IL-1 may both play an important role in the fatal outcome of gram-negative sepsis [19]. The inhibition of the LPS-induced plasma levels of these cytokines by a concomitant administration of bikunin is, therefore, expected to render mice less susceptible to a lethal dose of LPS.
We then investigated whether HI-8 was able to suppress the LPS-mediated up-regulation of cytokine expression. However, HI-8 did not suppress the LPS-stimulated up-regulation of the expression of TNF-, IL-1, and IL-6 at concentrations as high as 100 mg/kg.
Similar LPS-induced up-regulation of cytokine expression in macrophages isolated from Bik-/- and wt mice.
To assess whether differences in the expression of endogenous bikunin could account for the differences in the plasma concentration of cytokines observed in the in vivo study, we investigated, in an in vitro experiment, whether the LPS-induced up-regulation of TNF- expression was affected in macrophages isolated from Bik-/- mice. We used peritoneal macrophages to investigate reasons for the higher levels of TNF- in the plasma of Bik-/- mice after the administration of LPS [14]. We determined the cytokine levels in medium from macrophages after LPS challenge. LPS stimulated TNF- synthesis in wt macrophages in a dose- and time-dependent manner (data not shown). Bik-/- macrophages produced TNF- in the same amounts as wt macrophages when they were stimulated with LPS at 100 ng/mL for 9 h (figure 4A). Thus, the LPS-induced up-regulation of TNF- expression was not enhanced in Bik-/- macrophages that lacked endogenous bikunin.
In a parallel experiment, macrophages isolated from either Bik-/- or wt mice preincubated with bikunin-depleted FCS were treated with LPS for 9 h. Bikunin depletion resulted in an up-regulation of TNF- expression after LPS stimulation (figure 4A). LPS had similar effects on macrophages isolated from wt and Bik-/- mice. These results strongly suggest a role of bikunin in the suppression of the LPS-induced up-regulation of TNF- expression. It has been established that plasma derived from Bik-/- mice does not contain proteins of the II family, including bikunin [13, 15]. Taken together, these data suggest that plasma II, including bikunin, may play an important role in the suppression of the LPS-induced up-regulation of cytokine expression.
In a parallel experiment, IL-1 was induced in macrophages in response to LPS (not shown). This induction was also similar in Bik-/- mice, compared with that in wt mice. Both wt and Bik-/- macrophages responded similarly to bikunin, with the IC50 for the inhibition of IL-1 secretion being 1 mol/L in both wt and Bik-/- cells. These results suggest that bikunin plays an important role in regulating the sensitivity of macrophages to LPS-mediated cytokine expression.
Activation of ERK1/2, JNK, and p38 in macrophages from Bik-/- and wt mice.
LPS activation requires the phosphorylation of ERK1/2, JNK, and p38 in macrophages [20]. To determine whether LPS could activate ERK1/2, JNK, and p38 to the same extent in wt and Bik-/- macrophages, these cells were stimulated with LPS (100 ng/mL), and the lysates were immunoblotted with antiphosphoprotein antibodies. This resulted in the rapid (within 5 min) phosphorylation of ERK1/2, JNK, and p38, which peaked at 15 min and was still apparent after 30 min (figure 5A). The phosphorylation of ERK1/2, JNK, and p38 was not altered in Bik-/- macrophages (figure 5A, right), compared with wt macrophages (figure 5A, left). These data demonstrate that the LPS-induced phosphorylation of ERK1/2, JNK, and p38 is comparable between wt and Bik-/- macrophages and suggest that endogenous bikunin in macrophages does not play a critical role in the LPS-induced up-regulation of TNF-, at least through the activation of ERK1/2, JNK, and p38.
To investigate whether LPS-mediated signaling is also regulated by exogenous bikunin, we examined the effect of bikunin on LPS-stimulated signaling activation. The cells pretreated with 05 mol/L bikunin for 1 h were treated with LPS for 15 min, and then the phosphorylated bands were analyzed by Western blot (figure 5B). The ERK1/2 phosphorylation in both Bik-/- and wt cells was down-regulated by bikunin in a dose-dependent manner. Thus, 1 mol/L bikunin was sufficient to suppress the activation of ERK1/2 in Bik-/- and wt cells. No difference was found in the suppression by bikunin of LPS-induced activation of ERK1/2 in Bik-/- mice, compared with wt mice.
In a parallel experiment, macrophages isolated from Bik-/- or wt mice and preincubated with bikunin-depleted FCS were treated with LPS for 15 min. Bikunin depletion caused activation of ERK1/2 after LPS stimulation (figure 5B). LPS had similar effects on macrophages isolated from both wt and Bik-/- mice. These data suggest that bikunin may play an important role in the suppression of LPS-induced activation of the ERK1/2 signaling pathway. Furthermore, basal and stimulated cytokine levels through the ERK1/2, JNK, and p38 signaling pathways in Bik-/- macrophages were found to be the same as those in the wt mice (data not shown). These data allow us to speculate that the increased sensitivity of Bik-/- mice to LPS-induced death in vivo is due to a lack of bikunin in plasma.
DISCUSSION
Endotoxic shock is a systemic inflammatory process that is characterized histologically by cell damage, tissue necrosis, and vascular disruption [21]. LPS activates inflammatory cells, causing them to synthesize and release signals and molecules that contribute to the pathophysiologic process of septic shock [22, 23]. TNF-, IL-1, and nitric oxide (NO) are particularly essential moleculesmice deficient in TNF-, TNF- receptors, IL-1converting enzyme, inducible NO synthase, and caspase-11 [24], which are required for the processing of IL-1, are markedly resistant to LPS-induced mortality [25, 26].
In the present study, we used bikunin-deficient mice, which exhibited significantly shorter survival against lethal doses of bacterial LPS, suggesting a potential involvement of the II and bikunin pathway in the progression and pathogenesis of endotoxic shock. We showed that II- and bikunin-deficient mice produce significantly more TNF- and IL-1 in response to LPS than do wt mice. In LPS-treated mice, the production of TNF- was detected in various tissuesincluding lung, spleen, kidney, liver, and uterus/fallopian tubes [27]where bikunin message strongly expressed [28].
We then investigated the mechanism that leads to the excessive increase in TNF- and IL-1 levels in Bik-/- macrophages, which lack endogenous bikunin. For this, we used peritoneally derived macrophages obtained from mice. First, we examined the hypothesis that bikunin can serve to down-regulate the synthesis of cytokine expression. Indeed, the LPS-induced production of TNF- and IL-1 was enhanced in macrophages treated with bikunin-depleted serum. Second, exogenously applied bikunin also decreased LPS-stimulated TNF- synthesis in Bik-/- and wt macrophages in a dose-dependent manner. Suppression by bikunin of the LPS-induced up-regulation of TNF- expression was similar in both types of macrophages. Therefore, bikunin may play a role in the regulation of cytokine synthesis to prevent inflammation, including endotoxic shock. The mechanism by which cytokine levels are decreased may involve the bikunin-dependent suppression of de novo protein synthesis as induced by LPS and/or the release from existing intra- and extracellular pools. Our in vitro and in vivo results demonstrate that exogenous bikunin, but not HI-8, may decrease the LPS-induced production of proinflammatory cytokines (TNF- and IL-1), probably by decreasing the synthesis of cytokines by macrophages. These data are in full accordance with our findings that bikunin altered the in vitro kinetics of the LPS-dependent activation of signal cascadesincluding ERK1/2, JNK, and p38and greatly decreased the phosphorylation of signaling molecules in macrophages in response to LPS. The absence of II and bikunin from the plasma (in bikunin-deficient mice) led to a higher sensitivity mice to treatment with LPS in these than in wt mice, because a 1.52.0-fold increase in TNF- and IL-1 levels was observed in Bik-/- mice. These findings suggest that proteins of the II family promote the survival of LPS-injected mice, possibly through the suppression of cytokine expression, which indicates that a deficiency of II and bikunin in plasma could increase susceptibility to endotoxic shock.
Our findings clearly show that exogenous bikunin plays a role in suppressing endotoxic shock in vivo. Additionally, the results of our in vitro experiments suggest that bikunin down-regulates the expression of some cytokines. Thus, we demonstrate, for the first time, the effects of bikunin depletion on LPS-induced mortality in knockout mice. On the other hand, because decreased systemic levels of II have been observed in patients with severe sepsis [11], circulating II might also play a role in suppressing the progression of inflammatory conditions. Recent studies have shown that the administration of II prolongs the survival of animals in a model of endotoxic shock [29], as well as of humans with severe sepsis [11, 30]. Thus, these findings suggest that, in addition to bikunin, II also plays a role in preventing endotoxic shock. Although there is no information about the II-dependent signaling pathway at present, the possibility that it is also involved in the progression of endotoxic shock through II binding to macrophages and/or via its protease inhibitory activity deserves attention. We cannot exclude the possibility that, after the intravenous injection of II, it may cleave to bikunin and to heavy chains. It was remarkable that deficiency of proteins of the II family significantly altered susceptibility to endotoxic shock. This finding will be important in investigating genetic factors that affect susceptibility to septic shock in patients.
Until recently, bikunin has been assigned a classical protease inhibitory role. Recent data indicate that bikunin may have other functions that are unrelated to protease inhibition. We have shown that bikunin binds to cancer cells and to macrophages to suppress a variety of biological responses, predominantly through the ERK1/2 signaling pathway. On the basis of our previous data, we showed that bikunin binds to certain cells via specific bikunin-binding sites [3, 4]. Cell-associated bikunin-binding proteins (bikunin-BPs) may be critical targets for bikunin, given that HI-8, which does not possess a ligand for bikunin-BPs [4], did not prevent the LPS-stimulated up-regulation of cytokine expression. Membrane-associated bikunin-BPs are believed to represent the rate-limiting step for bikunin-dependent signal transduction or the cellular uptake of the bikunin. Therefore, bikunin-dependent protease inhibitory activity may not be crucial in the reduction of LPS-induced up-regulation of cytokine expression or LPS-induced lethality in animals.
Bikunin carries a chondroitin sulfate chain to which the heavy chains are covalently linked. The heavy chains can be transferred from II to hyaluronan molecules and become covalently linked on the extracellular matrix. Thus, heavy chains could stabilize the extracellular matrix by cross-linking hyaluronan molecules. Heavy chains linked to hyaluronan molecules have also been found in inflamed tissues. However, the physiological role of heavy chains of the II is not known. Taken together, these results suggest that proteins of the II family, including bikunin, are anti-inflammatory agents.
In conclusion, our findings suggest that bikunin plays a role in suppressing endotoxic shock and that its protease inhibitory activity may not be crucial in the reduction of LPS-induced lethality in vivo. We postulate that, in severe gram-negative bacterial infection, plasma bikunin and/or II proteins form a defense mechanism against the development of sepsis. If septic shock is not adequately neutralized, the administration of bikunin concentrates may be of therapeutic significance in overcoming the failure of this endogenous defense mechanism. Study of the underlying mechanisms will aid our understanding of the function and signal transduction of bikunin in cytokine production during endotoxic shock. The bikunin-deficient mouse is a valuable tool for further elucidation of the in vivo role of bikunin in various disease conditions in which bikunin might be involved. Finally, the present results should be of great value for research on the development of cytokine productionblocking agents as therapeutic drugs for septic shock or severe inflammation.
Acknowledgments
We thank H. Morishita and H. Sato (BioResearch Institute, Mochida Pharmaceutical, Gotenba, Shizuoka), Y. Tanaka and T. Kondo (Chugai Pharmaceutical, Tokyo, Japan), and S. Miyauchi and M. Ikeda (Seikagaku Kogyo, Tokyo, Japan), for their continuous and generous support of our work.
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