Hydroxy acid-based matrix metalloproteinase (MMP) inhibitors have been shown to inhibit tumor infiltration and growth, endotoxin shock, and acute graft-versus-host disease. Blockade of the release of soluble tumor necrosis factor-α (TNF-α) and CD95 ligand (CD95L; FasL) from cell-associated forms is reportedly involved in the mechanism of the drug effect. We investigated the effect of a MMP inhibitor, KB-R7785, on host resistance against Listeria monocytogenes infection, in which TNF-α is essentially required for the defense, in mice. The administration of KB-R7785 exacerbated listeriosis, while the drug prevented lethal shock induced by lipopolysaccharide and d-galactosamine. KB-R7785 inhibited soluble TNF-α production in spleen cell cultures stimulated by heat-killed L. monocytogenes and the drug treatment reduced serum TNF-α levels in infected mice, whereas the compound was ineffective on the modulation of interferon-γ and interleukin-10 production. The effect of KB-R7785 was considered to be dependent on TNF-α because the drug failed to affect L. monocytogenes infection in anti-TNF-α monoclonal antibody-treated mice and TNF-α knockout mice. Anti-CD95L monoclonal antibody was also ineffective on the infection. These results suggest that induction of infectious diseases, to which TNF-α is critical in host resistance, should be considered in MMP inhibitor-treated hosts.
Tumor necrosis factor-α
Matrix metalloproteinases (MMPs) constitute a large family of proteinases that degrade basement membrane and connective tissues, and are believed to play a pivotal role in the invasion and metastasis of tumor cells [1–3]. Some hydroxamic acid-based MMP inhibitors have been shown to inhibit tumor infiltration and growth in experimental animal models or phase I/II clinical trials in pancreatic, prostatic, and ovarian cancer patients [4–7]. Moreover, these MMPs are involved in the shedding of multiple cell surface molecules such as tumor necrosis factor-α (TNF-α) [8–10] and CD95 ligand (CD95L; FasL) . These drugs are reportedly able to prevent lethality in mice caused by endotoxin shock or concanavalin A-induced hepatitis through inhibiting the processing of TNF-α[8–10,12].
KB-R7785, a hydroxamic acid-based MMP inhibitor, inhibits MMP1, MMP3, and MMP9 [13,14]. Hattori et al. [11,15,16] reported that KB-R7785 prevents acute graft-versus-host disease in mice. They demonstrated that the inhibition of the release of TNF-α and CD95L from the cell surface is involved in prevention by KB-R7785 of acute graft-versus-host disease.
Listeria monocytogenes, a facultative intracellular bacterium, induces TNF-α in infectious foci such as in spleen and liver in mice . In vivo administration of anti-TNF antibodies into mice abrogated host resistance against L. monocytogenes infection [18,19] and extremely high susceptibility to the pathogen was observed in TNF receptor type I, or TNF-α and lymphotoxin-α double knockout mice [20–22], suggesting that host resistance to L. monocytogenes infection is critically dependent on TNF. Similarly, TNF is involved in host resistance to other facultative intracellular bacteria including Mycobacterium tuberculosis, Salmonella enterica serovar Typhimurium , and Yersinia enterocolitica.
In this study, we focused on the possibility that the administration of a MMP inhibitor may induce exacerbation of infectious diseases by pathogens in which TNF-mediated host defense is critical. We investigated the effect of the MMP inhibitor KB-R7785 [11,13–16] on host resistance against L. monocytogenes infection. We report that the administration of the MMP inhibitor reduces host resistance against L. monocytogenes infection and that a decrease in soluble TNF-α production is involved in the effect of the drug. Moreover, we show that the generation of acquired antilisterial resistance is not inhibited by the drug treatment.
2 Materials and methods
Female C57BL/6 mice, 5–7 weeks old, were purchased from Clear Japan, Inc., Tokyo, Japan. TNF-α-deficient mice (TNF-α−/−) on a C57BL/6×Sv129 background  were used at the age of 5–7 weeks. All animals were maintained under specific-pathogen-free conditions in the Institute for Animal Experiment, Hirosaki University School of Medicine.
2.2 L. monocytogenes infection and lethal endotoxin shock
L. monocytogenes 1b-1684 cells were prepared as described previously . Mice were infected intravenously with 0.2 ml of a solution containing various numbers of viable L. monocytogenes in 0.01 M phosphate-buffered saline (PBS, pH 7.4). 50% lethal doses for C57BL/6 mice and TNF-α−/− mice were 5×105 colony-forming units (cfu) and 100 cfu, respectively. The number of L. monocytogenes in the spleens and livers of infected animals was established by plating serial 10-fold dilutions of organ homogenates in PBS on tryptic soy agar (Difco Laboratories, Detroit, MI, USA). Colonies were counted 24 h later. To induce lethal endotoxin shock, mice were injected intraperitoneally with 8 mg of d-galactosamine and 10 µg of Escherichia coli lipopolysaccharide (LPS) (Sigma Chemical Co., St. Louis, MO, USA).
2.3 MMP inhibitor
The hydroxamic acid-based metalloproteinase inhibitor [4-(N-hydroxyamino)-2R isobutyl-3S-methylsuccinyl]-l-phenylglycine-N-methylamide (KB-R7785) was used [13,14]. For in vitro studies, this compound was dissolved in 10 mM dimethylsulfoxide. For in vivo administration, this compound was suspended in 0.5% carboxymethyl cellulose (CMC) at 10 mg ml−1. 0.5% CMC vehicle was used as the control. Mice were injected subcutaneously with 2 mg per 0.2 ml of KB-R7785 or with 0.2 ml of 0.5% CMC .
2.4 Injection with monoclonal antibodies against TNF-α and CD95L
Hybridoma cell lines secreting monoclonal antibodies (mAbs) against mouse TNF-α (MP6-XT22.11; rat IgG1) and against mouse CD95L (MFL-1; hamster IgG)  were injected into pristane-primed CD-1 nu/nu mice. The mAbs found in the ascites fluid were partially purified by (NH4)2SO4 precipitation . The mice were given a single intravenous injection of 50 µg of anti-TNF-α mAb or 1 mg of anti-CD95L mAb 1 h before infection. Normal rat globulin or normal hamster globulin was injected as a control for anti-TNF-α mAb and anti-CD95L mAb, respectively.
2.5 Cytokine assays
For estimation of cytokine production in vitro, spleen cells which were prepared as described previously  were resuspended in RPMI 1640 medium (Gibco Laboratories, Grand Island, NY, USA) supplemented with 10% fetal calf serum, 100 U ml−1 of penicillin G, and 100 µg ml−1 of streptomycin and then placed in 24-well tissue culture plates (Greiner, Frickenhausen, Germany) at a cell density of 5×106 cells per well in a final volume of 1 ml. Heat-killed L. monocytogenes cells at a concentration of 5×107 cells per well were added to the spleen cells. After 48 h of incubation at 37°C in a humidified 5% CO2 incubator, the supernatants were collected. For estimation of endogenous cytokine production, the livers and spleens from infected mice were suspended in RPMI 1640 medium containing 1% (w/v) CHAPS (Wako Pure Chemical Co., Osaka, Japan) and 10% (w/v) homogenates were prepared with a Dounce grinder and then clarified by centrifugation at 2000×g for 20 min . The culture supernatants, organ extracts, and sera were stored at −80°C until cytokine assays were performed. TNF-α, interferon-γ (IFN-γγ#x03B3;), and interleukin-10 (IL-10) assays were carried out using double sandwich ELISAs as described previously . Purified hamster anti-mouse TNF-α mAb (Genzyme Co., Boston, MA, USA) and rabbit anti-recombinant mouse TNF-α serum (Genzyme) were used for TNF-α ELISA. Purified rat anti-mouse IFN-γγ mAb (R4-6A2) and rabbit anti-recombinant mouse IFN-γγ serum  were used for IFN-γγ ELISA. Rat anti-mouse IL-10 mAb (JES5-2A5, PharMingen, San Diego, CA, USA) and biotinylated rat anti-mouse IL-10 mAb (SXC-1, PharMingen) were used for IL-10 ELISA.
2.6 Statistical evaluation of the data
Data were expressed as mean±S.D. and the Wilcoxon rank sum test was used to determine the significance of the differences of bacterial counts in the organs and the cytokine titers between the control and experimental groups. The generalized Wilcoxon test was used to determine the significance of differences in the survival rates. Each experiment was repeated at least three times and accepted as valid only when the trials showed similar results.
3.1 KB-R7785 blocks lethal endotoxin shock in mice
To confirm the preventive effect of KB-R7785 on lethal endotoxin shock, mice were injected subcutaneously with 2 mg of KB-R7785 or 0.5% CMC vehicle 1 h before the induction of shock by injection of LPS and d-galactosamine of endotoxin. More than 80% of the 0.5% CMC-injected control mice undergoing endotoxin shock died within 24 h, whereas most of the mice receiving KB-R7785 survived (Fig. 1).
Effect of a MMP inhibitor on lethal endotoxin shock in mice. C57BL/6 mice were injected subcutaneously with KB-R7785 in 0.5% CMC or 0.5% CMC vehicle 1 h before the induction of endotoxin shock by injection with LPS and d-galactosamine. The survival of each group was observed for 7 days. Twenty-one drug-injected mice and 20 control mice were used in three experiments. *Significant difference from the control group at P<0.01.
3.2 KB-R7785 inhibits host resistance against L. monocytogenes infection
It was investigated whether KB-R7785 might modulate host resistance against L. monocytogenes infection. Mice were infected with 5×105 cfu of L. monocytogenes. KB-R7785 or 0.5% CMC vehicle was injected into the mice 24 h and 1 h before infection and every 24 h until the end of observation of the survival of each group. In contrast to the control mice, KB-R7785-treated mice began to die on day 3 of infection and more than 60% of the mice finally succumbed (Fig. 2). Next, it was investigated whether KB-R7785 treatment might affect the number of L. monocytogenes cells in the organs. Mice were infected with 5×104 cfu of L. monocytogenes, which causes a nonlethal infection, or 5×106 cfu, which causes a lethal infection. KB-R7785 or the control vehicle was injected into the mice 24 h and 1 h before infection and every 24 h until the end of the experiment. Bacterial numbers in the livers and spleens of KB-R7785-treated mice were significantly higher than those of the controls in nonlethal infection (Fig. 3A,B). Similarly, the bacterial growth in the organs was notably augmented in KB-R7785-treated mice in lethal infection (Fig. 3C,D).
Effect of a MMP inhibitor on survival rates of mice infected with a sublethal dose of L. monocytogenes. Twenty C57BL/6 mice were injected with KB-R7785 or 0.5% CMC vehicle 24 h and 1 h before infection with 5×105 cfu of L. monocytogenes. KB-R7785 or the control vehicle was injected every 24 h until observation of the survival of each group was stopped. *Significant difference from the control group at P<0.01.
Effect of a MMP inhibitor on the bacterial growth in the livers (A,C) and spleens (B,D) of mice infected with a nonlethal dose or a lethal dose of L. monocytogenes. C57BL/6 mice were injected with KB-R7785 or 0.5% CMC vehicle 24 h and 1 h before infection with 5×104 cfu (A,B) or 5×106 cfu (C,D) of L. monocytogenes. The number of L. monocytogenes cells in the organs was determined on the indicated days. KB-R7785 or the control vehicle was injected every 24 h until the experiment was ended. Each point represents the mean±S.D. for a group of five mice in nonlethal infection and for a group of nine mice in lethal infection except four mice on day 4 in lethal infection. Significant differences from the control group: *P<0.01, * *P<0.05.
3.3 KB-R7785 inhibits the release of soluble TNF-α
The previous study demonstrated that KB-R7785 blocks the release of TNF-α from the cell surfaces . To confirm that KB-R7785 would reduce the production of soluble TNF-α induced by L. monocytogenes, spleen cells obtained from naive mice were stimulated with heat-killed L. monocytogenes cells in the presence of various concentrations of KB-R7785 dissolved in 10 mM dimethylsulfoxide. After cultivation for 48 h, the titers of TNF-α, IFN-γγ, and IL-10 in the culture supernatants were determined (Fig. 4). The TNF-α titers were clearly decreased, depending on the concentrations of KB-R7785 added. IL-10 production was partially decreased in the presence of KB-R7785 but the decrease was not dependent on the dose of the drug. The MMP inhibitor showed no significant effect on the IFN-γγ titers. Moreover, we investigated the effect of KB-R7785 on the release of soluble TNF-α induced by L. monocytogenes in vivo. Mice were infected intravenously with 5×106 cfu of viable L. monocytogenes cells. KB R7785 or 0.5% CMC vehicle was injected into the mice 24 h and 1 h before infection and 24 h post infection. Titers of sera, spleen homogenates and liver homogenates at 48 h post infection were determined (Fig. 5). The serum TNF-α titers, which represent the content of soluble TNF-α, in KB-R7785-injected mice were significantly decreased, compared with that in the vehicle-treated mice (P<0.01), whereas the TNF-α titers of spleen and liver homogenates, which contain both soluble and cell-associated TNF-α, were not affected by KB-R7785 treatment.
Effect of a MMP inhibitor on the release of cytokines into the media of spleen cell cultures. Spleen cells obtained from C57BL/6 mice were stimulated with heat-killed L. monocytogenes cells in the presence of various concentrations of KB-R7785 dissolved in 10 mM dimethylsulfoxide. After cultivation for 48 h, the titers of TNF-α, IFN-γγ, and IL-10 in the culture supernatants were determined.
Effect of a MMP inhibitor on endogenous TNF-α production induced by L. monocytogenes. Five C57BL/6 mice were injected with KB-R7785 or 0.5% CMC vehicle 24 h and 1 h before infection and 24 h post infection. They were infected with 5×106 cfu of L. monocytogenes. Blood, livers, and spleens were taken 48 h later and TNF-α titers of the sera (A), liver homogenates (B), and spleen homogenates (C) were determined. *Significant difference from the control group at P<0.01.
3.4 TNF-α dependence of KB-R7785 action
KB-R7785 reportedly inhibits MMP-mediated release of TNF-α and CD95L [11,15,16]. To investigate TNF-α dependence and implication of CD95L in KB-R7785 action, mice were injected with anti-TNF-α mAb 2 h before infection, and they were administered KB-R7785 or 0.5% CMC vehicle 24 h and 1 h before infection with 500 cfu of L. monocytogenes. Mice were injected with the drug every 24 h until the experiment was stopped. The number of L. monocytogenes cells in the organs was determined on day 3 of infection. The KB-R7785 treatment was ineffective on the bacterial growth in livers and spleens in anti-TNF-α mAb-treated mice (Fig. 6A). Similarly, TNF-α−/− mice were injected with KB-R7785 or 0.5% CMC vehicle 24 h and 1 h before infection with 100 cfu of L. monocytogenes. They were injected with the drug or CMC vehicle 24 h and 48 h later, and the number of L. monocytogenes cells in the organs was determined on day 3 of infection. Bacterial load in the organs was comparable between MMP inhibitor-treated mice and CMC-treated mice (Fig. 6B). Recent studies using CD95-defective MRL/lpr mice showed that the CD95-CD95L system is partially involved in antilisterial resistance [31–33]. To investigate the effect of anti-CD95L mAb on antilisterial resistance, TNF-α−/− mice were injected intravenously with 1 mg of anti-CD95L mAb or normal hamster globulin 1 h before infection with 100 cfu of L. monocytogenes. Numbers of L. monocytogenes cells in the organs on day 3 of infection were as follows: normal hamster globulin-treated mice versus anti-CD95L mAb-treated mice (log10 bacteria±S.D., n=5 each): 6.18±0.10 versus 6.28±0.14 in the livers (P>;0.05) and 5.91±0.33 versus 5.69±0.88 in the spleens (P<0.05).
Effect of a MMP inhibitor on the growth of L. monocytogenes cells in the organs of mice in the absence of TNF-α. C57BL/6 mice receiving anti-TNF-α mAb (A) and TNF-α−/− mice (B) were infected with 500 cfu or 100 cfu of L. monocytogenes, respectively. They were injected with KB-R7785 or 0.5% CMC vehicle 24 h and 1 h before infection. The number of L. monocytogenes cells in the organs was determined on day 3 of infection. KB-R7785 or the control vehicle was injected every 24 h until the experiment was ended. Each data point represents the mean±S.D. for a group of five mice.
3.5 KB-R7785 fails to modulate the generation of acquired immunity
L. monocytogenes promotes the induction of the T-helper 1 (Th1) response, including IFN-γγ production , and IFN-γγ is another critical factor in antilisterial resistance . It was investigated whether KB-R7785 might modulate the induction of the Th1 response by L. monocytogenes infection. Mice were injected with KB-R7785 or 0.5% CMC vehicle 24 h and 1 h before infection with 5×104 cfu of L. monocytogenes, and then the drug was injected every 24 h until day 5 of infection. Spleen cells, which were obtained from KB-R7785-injected mice and 0.5% CMC-treated mice on day 8 of infection, were stimulated with heat-killed L. monocytogenes cells. IFN-γγ titers in 48-h culture supernatants were determined. No differences in the IFN-γγ titers were observed between two groups: KB-R7785-treated mice versus 0.5% CMC-treated mice (ng ml−1±S.D., n=4): 8.45±0.46 versus 8.85±0.04. Next, it was investigated whether KB-R7785 might modulate the generation of an acquired resistance against L. monocytogenes infection. Mice were injected with KB-R7785 or 0.5% CMC vehicle 24 h and 1 h before infection with 5×104 cfu of L. monocytogenes, and then the drug was injected every 24 h until day 5 of infection. On day 10 of infection, they were challenged with 5×106 cfu of L. monocytogenes. The number of bacterial cells in the livers and spleen was determined 48 h later (Table 1). The elimination of bacteria was augmented in the immunized mice. However, bacterial numbers in the organs were not affected by KB-R7785 treatment.
Effect of KB-R7785 on acquired antilisterial immunity
Log number of bacteria/organ
↵C57BL/6 mice were immunized i.v. with 5×104 cfu of L. monocytogenes. They were injected with KB-R7785 or 0.5% CMC 24 h and 1 h before immunization. On day 10 of immunization, they were challenged i.v. with 5×106 cfu of L. monocytogenes and the number of bacterial cells in the organs was determined 48 h later.
It has been demonstrated that host resistance against L. monocytogenes infection is critically dependent on TNF, especially TNF-α[18–22]. Therefore, it is possible that hydroxamic acid-based MMP inhibitors, which inhibit the processing of TNF-α, may regulate host resistance against L. monocytogenes infection. To address it, we investigated the effect of a hydroxamic acid-based MMP inhibitor, KB-R7785, on host resistance against L. monocytogenes infection. This study demonstrated that the MMP inhibitor suppresses antilisterial resistance.
Hydroxamic acid-based MMP inhibitors are known to be able to prevent lethality in mice caused by endotoxin shock or concanavalin A-induced hepatitis through inhibiting the processing of TNF-α[8,12]. We confirmed the preventive effect of KB-R7785 on lethal endotoxin shock (Fig. 1). TNF-α was a critical factor in lethality because anti-TNF-α mAb-treated mice and TNF-α−/− mice were rescued from the lethal shock in our study (data not shown). In contrast, the mortality was accelerated by KB-R7785 treatment in sublethal L. monocytogenes infection (Fig. 2) and it was due to the reduced ability to eliminate L. monocytogenes from the organs (Fig. 3). A MMP inhibitor reportedly exacerbates liver injury in concanavalin A-induced hepatitis through the induction of hepatocellular necrosis and apoptosis by cell-associated TNF-α and CD95L [12,36]. However, apoptosis in the livers of L. monocytogenes-infected mice was not augmented by KB-R7785 treatment (Yamada et al., unpublished observation).
Previous studies have demonstrated that KB-R7785 inhibits the cleavage of cell-associated TNF-α. In the present study, KB-R7785 reduced the release of TNF-α into the culture medium from spleen cells stimulated with L. monocytogenes (Fig. 4). The administration of KB-R7785 reduced serum TNF-α titers in L. monocytogenes infection in vivo (Fig. 5). However, the MMP inhibitor showed no significant effect on L. monocytogenes-induced IFN-γγ production, which is performed mainly by natural killer cells , and IL-10 production, which is performed by macrophages like TNF-α (Fig. 4), suggesting that KB-R7785 does not interfere with the production of cytokines such as IFN-γγ and IL-10. On the other hand, KB-R7785 did not inhibit endogenous TNF-α production in the spleens and livers of L. monocytogenes-infected mice, which contain both soluble and cell-associated TNF-α (Fig. 5). These results suggest that KB-R7785 may not inhibit TNF-α production but it may block the processing of TNF-α.
It was demonstrated that CD95L, which is a type II integral membrane protein homologous with TNF-α, is shed by MMPs from activated T cells and that KB-R7785 prevented a lethal acute graft-versus-host disease through blocking the release of CD95L [11,15,16]. CD95-CD95L-mediated immunity is reportedly involved in host defense to L. monocytogenes infection, although perforin-dependent host defense could be complemented in the absence of the CD95-CD95L system, i.e. CD95-defective MRL/lpr mice were substantially resistant to L. monocytogenes infection [31–33]. Herein, we also showed that the administration of anti-CD95L mAb failed to affect antilisterial resistance in TNF-α−/− mice. Hence, the effect of KB R7785 on L. monocytogenes infection might be CD95L-independent.
Activated macrophages along with neutrophils are important effector cells in antilisterial resistance . Therefore, we assumed that KB-R7785 might inhibit the activation of phagocytes. However, the elimination of L. monocytogenes cells from spleen and liver was augmented in the mice which were injected with LPS irrespective of KB-R7785 treatment (data not shown). It has been demonstrated that both IFN-γγ and TNF-α play critical roles in antilisterial resistance and that one of their roles is the activation of macrophages [19,35]. KB-R7785 showed no inhibitory effect on the production of IFN-γγ (Fig. 4), suggesting that KB-R7785 might not interfere with the activation of phagocytes. Subsequently, Th1 and CD8+ cytolytic T lymphocytes are reportedly essential for the complete elimination of L. monocytogenes cells from organs in both primary and secondary infections . Herein, KB-R7785 treatment inhibited the induction of acquired antilisterial immunity (Table 1).
KB-R7785 also inhibits activities of different MMPs such as MMP1, MMP3, and MMP9 [13,14]. MMPs are reportedly involved in the development of organ damage in endotoxemia . In this study, the dramatic effect of KB-R7785 on histological changes of spleen and liver was not observed in L. monocytogenes-infected mice (data not shown). However, effects of the MMP inhibitor on the balance of MMPs and inhibitors of MMPs should be taken into consideration.
Finally, our present study suggests that induction of infectious diseases, to which TNF-α is critical in host resistance, should be considered in MMP inhibitor-treated hosts.
This work was supported in part by a grant-in-aid for general scientific research (08670297 and 10670247) provided by the Japanese Ministry of Education, Science, Sports and Culture.
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