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Originally published online as doi:10.1189/jlb.1006598 on May 23, 2007

Published online before print May 23, 2007
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(Journal of Leukocyte Biology. 2007;82:327-334.)
© 2007 by Society for Leukocyte Biology

Induction of serum IL-18 with Propionibacterium acnes and lipopolysaccharide in phagocytic macrophage-inactivated mice

Takashi Nishioka*,{dagger}, Toshinobu Kuroishi*, Yumiko Sugawara{dagger}, Zhiqian Yu{dagger}, Takashi Sasano{dagger}, Yasuo Endo* and Shunji Sugawara*,1

* Division of Oral Immunology, Department of Oral Biology, and
{dagger} Division of Oral Diagnosis, Department of Oral Medicine and Surgery, Tohoku University Graduate School of Dentistry, Sendai, Japan

1 Correspondence: Division of Oral Immunology, Department of Oral Biology, Tohoku University Graduate School of Dentistry, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan. E-mail: sugawars{at}mail.tains.tohoku.ac.jp


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ABSTRACT
 
IL-18, an important regulator of immune responses, is expressed in activated macrophages and also in nonimmune cells, such as keratinocytes and epithelial cells. Increased levels of serum IL-18 are reported in patients with a wide variety of diseases, but it is unclear which type of cell is the major source of serum IL-18. Here, we showed that the administration of liposomes encapsulating clodronate (Clo-lip) in mice selectively depleted F4/80+ phagocytic macrophages in the liver and spleen. Serum levels of mature IL-18 with 18 kDa were increased markedly in mice treated with Propionibacterium acnes and LPS, whereas administration of Clo-lip and gadolinium chloride, another widely used macrophage inactivator, showed no obvious effect on serum IL-18 levels, which were marginal in the liver, lung, and spleen and more pronounced in the intestines, especially in the duodenum. Treatment with P. acnes alone induced IL-18 more than twofold in each organ, and P. acnes and LPS induced a marked increase in IL-18 levels in the liver and spleen but decreased in the intestines. The administration of Clo-lip showed only a marginal effect on the IL-18 levels in these organs. Furthermore, serum levels of liver enzymes and TNF-{alpha} and liver injury (necrotic change and granuloma formation) induced by P. acnes and LPS were reduced moderately by Clo-lip. These results suggest that phagocytic macrophages do not actively contribute to the induction of serum IL-18 and liver injury in mice treated with P. acnes and LPS.

Key Words: cytokine • inflammation • macrophages • mucosal cells


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INTRODUCTION
 
IL-18 was identified originally as an IFN-{gamma}-inducing factor from a murine liver cell cDNA library generated from mice primed with heat-killed Propionibacterium acnes and subsequently challenged with LPS [1 ]. IL-18 is produced intracellularly as an inactive, 24-kDa precursor form (proIL-18) and secreted as an 18-kDa mature form after cleavage by caspase-1, originally designated IL-1β-converting enzyme [2 3 4 ]. IL-18 is now recognized as a multifunctional regulator of innate and acquired immune responses through its activation of Th1 and Th2 responses [2 3 4 5 ]. IL-18 has also been suggested to be a potent, proinflammatory cytokine, which regulates autoimmune and inflammatory diseases [2 3 4 ].

Recent studies showed that IL-18 is identified, not only in activated macrophages, including dendritic cells (DC) and Kupffer cells, but also in nonimmune cells, such as keratinocytes, osteoblasts, adrenal cortex cells, epithelial cells of various organs and tissues, microglial cells, and synovial fibroblasts [2 3 4 ]. This wide range of distribution implies that IL-18 plays physiological roles and acts as a component of immune regulation.

Increased levels of IL-18 have been reported in the sera from patients with a wide variety of diseases, including autoimmune and inflammatory disorders [6 7 8 9 10 11 ], allergy [12 ], allograft rejection [13 ], and infectious diseases [14 15 16 ], and the elevated serum IL-18 levels are considered to be a parameter for the disease severity and a diagnostic marker. We have shown recently that human oral epithelial cells constitutively express a precursor form of IL-18, stimulation of the cells with neutrophil proteinase 3 (PR3) and LPS induces the secretion of an active form of IL-18 after IFN-{gamma} priming [17 ], and PR3 activates the cells through a G protein-coupled protease-activated receptor 2 (PAR2) on the cell surface in vitro [18 , 19 ]. Subsequently, we also revealed that neutrophil recruitment and PAR2 activation are critically involved in the induction of serum IL-18 in mice treated with heat-killed P. acnes and LPS in vivo [20 ].

It is still unclear whether the major source of serum IL-18 is activated macrophages or nonimmune cells, such as those of epithelial origin in vivo. A macrophage "suicide" technique, using liposomes encapsulating dichloromethylene bisphosphonate (clodronate), specifically depletes phagocytic macrophages but not neutophils and DC within 1 day or 2 of the i.v. injection of such liposomes into mice or rats [21 22 23 ]. It is also reported that i.v. injection of gadolinium chloride (GdCl3) not only blocks phagocytosis of Kupffer cells but also eliminates these cells [24 , 25 ]. These techniques have allowed us to investigate whether the major source of serum IL-18 is activated macrophages in mice treated with P. acnes and LPS. We also examined the effect of macrophage inactivation by the liposomes on liver injury in the mice.


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MATERIALS AND METHODS
 
Mice
Female C57BL/6 mice (6–9 weeks old), obtained from the Institute for Experimental Animals of the Tohoku University Graduate School of Medicine (Sendai, Japan), were used for the experiments.

Bacteria and reagents
P. acnes was grown in brain-heart infusion medium (Difco Laboratories, Detroit, MI, USA) with L-cysteine and Tween-80, as described previously [20 ]. The harvested bacteria were washed with sterile, distilled water, killed by heating at 60°C for 1 h, and then lyophilized. The lyophilized bacteria were next suspended in PBS (5 mg/ml) and used to prime the mice. LPS from Escherichia coli O55:B5 and clodronate were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mouse recombinant (mr)IL-18 was obtained from Medical and Biological Laboratories (Okayama, Japan). Rabbit anti-mouse IL-18 polyclonal antibody (pAb) was provided by Tomaki Hoshino (Kurume University, Kurume, Japan). All other reagents were obtained from Sigma-Aldrich, unless otherwise indicated.

Treatment of mice with clodronate-liposomes (Clo-lip) or GdCl3
A suspension of liposomes encapsulating clodronate was prepared according to a method described previously [22 , 23 ]. Briefly, 75 mg phosphatidylcholine and 11 mg cholesterol were dissolved in chloroform (20 ml) in a round-bottomed flask (1000 ml). The thin film, which formed on the walls of the flask after rotary evaporation at 37°C, was dispersed by gentle shaking for 10 min in 10 ml clodronate solution (200 mg/ml) in PBS. This suspension was kept for 2 h at room temperature, then sonicated for 3 min (50 Hz), and kept for another 2 h. The resulting liposomes floating on the aqueous phase were collected using a Pasteur pipette, suspended in 10 ml PBS, and centrifuged at 5000 g for 30 min. The precipitated liposomes were finally suspended in 4 ml PBS; this preparation is referred to as the "original" suspension of Clo-lip. This original suspension was diluted (as described in the text) using PBS, and the diluted suspension was injected i.v. at 0.2 ml/mouse.

The mice were also injected i.v. with GdCl3 (10 mg/kg) or saline [25 ].

Histological analysis
Immunohistochemistry was conducted as follows. Tissues were fixed in periodate-lysine-4% paraformaldehyde for 6 h at 4°C. After washing in PBS containing sucrose, fixed tissues were embedded in OCT compound (Sakura, Tokyo, Japan) and frozen immediately. Frozen tissue sections (6 µm) were incubated with rat anti-mouse F4/80 mAb (Serotec, Oxford, UK) overnight at 4°C. After that, the sections were treated with peroxidase-blocking reagent (Dako Cytomation, Tokyo, Japan) for 20 min and secondary antibodies, such as the goat anti-rat simple-stain mouse MAX-PO (Nichirei, Tokyo, Japan). The chromogen used was 3',3-diaminobenzidine tetrahydrochloride (Dako Cytomation). The sections were counterstained with hematoxylin. As a negative control, rat isotype-matched control Ig G2b (BD Biosciences, San Diego, CA, USA) was used.

For histopathological analysis, formalin-fixed samples were embedded in paraffin and stained with H&E.

Treatment of mice and preparation of serum and tissue extracts
The mice were injected i.p. with heat-killed P. acnes (1 mg dry weight/mouse), and 7 days later, they were challenged i.v. with LPS (1 µg/mouse). As P. acnes-primed mice started to die of endotoxin shock 4 h after the LPS challenge [20 ], blood and tissues were taken from the mice 2 h after the LPS challenge in this study. Blood was collected directly into test tubes following their decapitation, and the serum was recovered by centrifugation at 2000 g at 4°C, after which it was stored at –80°C until use. Frozen tissues or organs were homogenized in RPMI 1640 containing Triton X-100 (5 µl/ml), HEPES (10 µmol/ml), BSA (100 µg/ml), gentamicin sulfate (50 µg/ml), and proteinase inhibitor cocktail (10 µl/ml) [26 ]. The cocktail contains 4-(2-aminoethyl)benzenesulfonyl fluoride, aprotinin, leupeptin, bestatin, pepstatin A, and E-64. The supernatants obtained by centrifugation at 10,000 g for 10 min at 4°C of the homogenates were then stored at –80°C until use. The experimental procedure followed in this study was approved by the Ethical Board for Non-Human Species of the Tohoku University Graduate School of Medicine (Sendai, Japan).

Measurement of cytokines and liver enzymes
The levels of IL-18 and TNF-{alpha} in the samples were determined using a mouse IL-18 ELISA kit (Medical and Biological Laboratories, Woburn, MA, USA) and a mouse TNF-{alpha} OptEIA ELISA kit (BD PharMingen, San Diego, CA, USA), respectively. According to the manufacturer, the IL-18 ELISA kit mainly detects an 18-kDa, mature form, and the sensitivity to the precursor form was less than 10% compared with the mature form. The amount of IL-18 in each tissue was expressed as µg/g wet tissue.

Serum asparate aminotransferase (AST) and alanine aminotransferase (ALT) activities were measured photometrically using commercial kits (Wako Pure Chemical Industries, Osaka, Japan).

Western blotting
All samples were solubilized with Laemmli sample buffer [27 ]. SDS-PAGE was performed in a 15% polyacrylamide slab gel under reducing conditions, according to the method of Laemmli [27 ]. Proteins were transferred to a polyvinylidene difluoride membrane using a semidry transblot system (Atto Instruments, Tokyo, Japan). The blot was blocked for 2 h with 3% w/v nonfat dry milk and 0.05% Tween 20 in PBS (Blotto/Tween) and incubated with anti-mouse IL-18 pAb at 6 µg/ml in Blotto/Tween overnight at 4°C. The blot was washed four times with Blotto/Tween and then incubated for 90 min with HRP-conjugated, affinity-purified goat anti-rabbit IgG at 1:3000 (Pierce Biotechnology, Rockford, IL, USA) in Blotto/Tween. After being washed, IL-18 was visualized with West Femto maximum sensitivity substrate (Pierce Biotechnology). The molecular weight of the proteins was estimated by comparison with the position of a standard (Bio-Rad Laboratories, Hercules, CA, USA).

Statistical analysis
Experimental values were expressed as the mean ± SD, and the statistical significance of differences between two means was evaluated by one-way ANOVA using the Bonferroni or Dunnett method, for which values of P < 0.05 were considered to be statistically significant.


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RESULTS
 
Effect of macrophage inactivation on the induction of serum IL-18 in mice
As it is suggested that activated macrophages, such as Kupffer cells, are the major source of serum IL-18 in vivo [1 , 2 ], we examined the effect of macrophage inactivation by Clo-lip or GdCl3 on the induction of serum IL-18 in mice after treatment with P. acnes and LPS. Clo-lip was administered in mice on Days 0, 3, and 5, and the livers and spleens were taken on Day 8. GdCl3 was administered in mice on Days 0, 4, and 7, and the livers and spleens were taken on Day 9. The results from immunohistochemistry showed that the administration of Clo-lip eliminated F4/80+ macrophages (Kupffer cells) in the liver (Fig. 1A ). F4/80+ macrophages were abundant in the red pulp of the spleen, and the Clo-lip treatment also eliminated F4/80+ macrophages in the spleen (Fig. 1B) . In contrast, GdCl3 did not deplete F4/80+ macrophages in the liver and spleen. These results indicate that Clo-lip efficiently eliminates phagocytic macrophages in mice; however, it remains possible that GdCl3 inhibits the functions of macrophages, even though it did not eliminate them [24 ]. Therefore, we used Clo-lip and GdCl3 in the next experiments.


Figure 1
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  		Figure 1. Effect of Clo-lip and GdCl3 on the depletion of macrophages in mice. Saline (Control) or Clo-lip (fivefold-diluted, 0.2 ml/mouse) was administered i.v. in C57BL/6 mice on Days 0, 3, and 5, and the livers and spleens were taken on Day 8. GdCl3 (10 mg/kg) was administered i.v. in C57BL/6 mice on Days 0, 4, and 7, and the livers and spleen were taken on Day 9. The liver (A) and spleen (B) were subjected to immunostaining with F4/80 antibody and visualized using diaminobenzidine (brown). The results are representative of three mice. Original scale bars, 200 µm.

Serum IL-18 levels were increased markedly by treatment with P. acnes and LPS, whereas the administration of Clo-lip, three times (1 day before and 2 and 4 days after P. acnes injection), or GdCl3, three times (2 days before and 3 and 5 days after P. acnes injection), showed no obvious effect on the serum IL-18 levels (Fig. 2A ). Consistent with this, Western blot analysis showed that an 18-kDa, mature form of IL-18 was detected in the sera of P. acnes and LPS-treated mice and that the band was unchanged by Clo-lip or GdCl3 treatment (Fig. 2B) . These results indicate that activated macrophages are not critically involved in the induction of serum IL-18 in mice treated with P. acnes and LPS and suggest that other cells are the source of serum IL-18.


Figure 2
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  		Figure 2. Effect of Clo-lip and GdCl3 on the induction of serum IL-18 in mice. (A) P. acnes (1 mg dry weight/mouse) or PBS was administered i.p. to C57BL/6 mice, and 7 days later, they were challenged i.v. with LPS (1 µg/mouse) or PBS. Clo-lip (fivefold-diluted, 0.2 ml/mouse) was administerted i.p. into the mice 1 day before and 2 and 4 days after P. acnes administration. GdCl3 (10 mg/kg) was administerted i.v. into the mice 2 days before and 2 and 5 days after P. acnes administration. Blood was then taken from the mice 2 h after LPS challenge, and the levels of IL-18 in the sera were determined by ELISA. The results were expressed as the means ± SD for five mice. **, P < 0.01, compared with untreated mice. (B) Samples in A were subjected to Western blotting using anti-mouse IL-18 pAb. mrIL-18 (1 ng) was loaded as a control. The results are representative of five mice of each group.

As Clo-lip and GdCl3 showed the same effect on the induction of serum IL-18 levels, and Clo-lip efficiently eliminated F4/80+ macrophages in mice, we used Clo-lip in the following experiments.

IL-18 levels in various organs in mice
We then examined the IL-18 levels in various organs in mice. In untreated, control mice, the IL-18 levels were marginal in the liver, lung, and spleen and more pronounced in the intestines (duodenum, jejunum, ileum, and colon), especially so in the duodenum (Fig. 3A ). Treatment with P. acnes alone induced IL-18 more than twofold in each organ except for the lung and colon. Treatment with P. acnes and LPS induced a marked increase in IL-18 levels in the liver and spleen but decreased them slightly in the duodenum, jejunum, and ileum, compared with those with P. acnes alone.


Figure 3
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  		Figure 3. IL-18 and levels in various organs or tissues in mice. (A) P. acnes (1 mg dry weight/mouse) or PBS was administered i.p. to C57BL/6 mice, and 7 days later, they were challenged i.v. with LPS (1 µg/mouse) or PBS. Organs or tissues were then taken from the mice 2 h after LPS challenge, and the levels of IL-18 in the samples were determined by ELISA. The results were expressed as the means ± SD for five mice. *, P < 0.05, and **, P < 0.01, compared with untreated mice. (B) Samples in A were subjected to Western blotting using anti-mouse IL-18 pAb. mrIL-18 (10 ng) was loaded as a control. The results are representative of five mice of each group.

Western blot analysis showed that proIL-18 was expressed at a low amount in the livers of untreated mice, and P. acnes treatment induced proIL-18 and mature IL-18 in the liver (Fig. 3B) . Treatment with P. acnes and LPS resulted in a further increase of both forms of IL-18. The spleens of untreated mice expressed proIL-18 and a band lower than 18 kDa, suggesting that mature IL-18 is not expressed in the untreated spleen. The expression of proIL-18 and mature IL-18 was induced by P. acnes alone and increased further by P. acnes and LPS in the spleen. In contrast, both forms existed already in the untreated duodenum, and mature IL-18 was increased markedly in the duodenum by P. acnes alone and by P. acnes and LPS. In the jejunum and ileum, mature IL-18 was mainly expressed, and the expression was increased and decreased slightly by P. acnes alone and P. acnes and LPS, respectively, compared with the untreated control. In the colon, the expression of pro-IL-18 was higher than that of mature IL-18 in the untreated control, and P. acnes treatment reduced pro-IL-18 and increased mature IL-18 expressions. The expression of proIL-18 was increased by P. acnes and LPS, compared with P. acnes alone. These results indicate that P. acnes priming in mice leads to the accumulation of mature IL-18 in these organs and renders the mice susceptible to LPS, that LPS challenge induced the release of mature IL-18, and that the diverse expression of pro- and mature IL-18 occurs in each organ and even in the intestines.

Effect of macrophage depletion on the IL-18 levels in organs in mice
As Figure 2 illustrates that Clo-lip showed no effect on serum IL-18 levels, the effect of Clo-lip on the IL-18 levels in various organs in mice was then examined. Treatment of mice with P. acnes and LPS markedly increased the IL-18 levels in the liver and spleen but not in the lung, and the administration of Clo-lip, three times (1 day before and 2 and 4 days after P. acnes injection) in the mice, slightly, but not significantly, decreased in the IL-18 levels in the liver (Fig. 4A ). However, a substantial amount of IL-18 remained in the liver following the Clo-lip treatment. In addition, the IL-18 levels in the spleens of mice treated with P. acnes and LPS did not change following the Clo-lip treatment. The IL-18 levels in the intestines are also unchanged by the Clo-lip treatment (data not shown). Consistent with this, Western blot analysis showed that the expression of mature IL-18 was almost unchanged in the livers and spleen of mice treated with P. acnes and LPS after the Clo-lip administration (Fig. 4B) . These results further indicate that IL-18 expressed in activated macrophages in organs and tissues does not cause the elevation of serum IL-18 levels.


Figure 4
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  		Figure 4. Effect of macrophage depletion on the IL-18 levels in the liver, lung, and spleen in mice. (A) P. acnes (1 mg dry weight/mouse) or PBS was administered i.p. to C57BL/6 mice, and 7 days later, they were challenged i.v. with LPS (1 µg/mouse) or PBS. Clo-lip (fivefold-diluted, 0.2 ml/mouse) was administered i.p. to the mice 1 day before and 2 and 4 days after P. acnes administration. The liver, lung, and spleen were then taken from the mice 2 h after LPS challenge, and the levels of IL-18 in the samples were determined by ELISA. The results are expressed as the means ± SD for five mice. **, P < 0.01, compared with untreated (Control) mice. (B) Samples in A were subjected to Western blotting using anti-mouse IL-18 pAb. The results are representative of five mice of each group.

Effect of macrophage depletion on LPS-induced liver injury in P. acnes-primed mice
As it has been reported that treatment with P. acnes and LPS induces IL-18-dependent, acute liver injury in mice through the induction of hepatotoxic factors such as TNF-{alpha} and that Kupffer cells are the major source of IL-18 in the liver [28 29 30 ], we next examined the effect of Clo-lip on the liver injury. The levels of the liver enzymes, AST and ALT, in sera were increased markedly by treatment with P. acnes and LPS, and their levels were decreased significantly by the three-time administration of Clo-lip (Fig. 5A ). However, the serum AST and ALT levels were still high compared with those of untreated mice. Treatment with P. acnes and LPS resulted in a marked increase in serum TNF-{alpha} levels, and the induction of serum TNF-{alpha} was slightly, but not significantly, reduced by Clo-lip treatment (Fig. 5B) . Consistent with this, histological analysis showed that treatment with P. acnes and LPS induced severe liver injury (necrotic change and granuloma formation) and cell infiltration, and treatment with P. acnes alone also induced granuloma formation and cell infiltration (Fig. 6 ). No granuloma was observed, and the cell infiltration was reduced by Clo-lip administration. However, the cell infiltration and necrotic change were still observed in the Clo-lip-administered liver. These results suggest that Kupffer cells are actually involved in liver injury in mice treated with P. acnes and LPS and that IL-18 and TNF-{alpha} from non-Kupffer cells also contribute to the liver injury.


Figure 5
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  		Figure 5. The effect of Clo-lip administration on serum levels of liver enzymes and TNF-{alpha}. (A) PBS or P. acnes (1 mg dry weight/mouse) was administered i.p. to C57BL/6 mice, and 7 days later, they were challenged i.v. with PBS or LPS (1 µg/mouse). Clo-lip (fivefold-diluted, 0.2 ml/mouse) was administered i.p. to the mice 1 day before and 2 and 4 days after P. acnes priming. Blood was taken from the mice 2 h after LPS or PBS challenge, and the serum AST and ALT levels were measured. (B) The levels of TNF-{alpha} in the samples in A were determined by ELISA. The results are expressed as the means ± SD for five mice. *, P < 0.05, and **, P < 0.01, compared with P. acnes and LPS.


Figure 6
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  		Figure 6. Effect of Clo-lip on LPS-induced liver injury in P. acnes-primed mice. PBS or P. acnes (1 mg dry weight/mouse) was administered i.p. to C57BL/6 mice, and 7 days later, they were challenged i.v., with PBS or LPS (1 µg/mouse). Clo-lip (fivefold-diluted, 0.2 ml/mouse) was administered i.p. to the mice 1 day before and 2 and 4 days after P. acnes priming. Liver specimens were then sampled 2 h after PBS or LPS challenge, and liver tissue sections were stained with H&E. The results are representative of five mice of each group. Original scale bars, 200 µm.


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DISCUSSION
 
IL-18 was identified originally as a potent IFN-{gamma}-inducing factor in the serum and livers of mice, which had been administered P. acnes and LPS sequentially [1 ]. IL-18 was first identified in activated macrophages, such as Kupffer cells, in the liver. Further investigations have revealed that IL-18 is also expressed in nonimmune cells, such as keratinocytes, osteoblasts, adrenal cortex cells, epithelial cells of various organs and tissues, microglial cells, and synovial fibroblasts [2 3 4 ]. Increased levels of IL-18 have been reported in the sera from patients with a wide variety of diseases, including autoimmune and inflammatory disorders [6 7 8 9 10 11 ], allergy [12 ], allograft rejection [13 ], and infectious diseases [14 15 16 ], and the elevated serum IL-18 levels are considered to be a parameter for the disease severity and a diagnostic marker. However, it is unclear whether the major source of serum IL-18 is immune cells, such as activated macrophages, or nonimmune cells, such as epithelial origin. The present study used Clo-lip to deplete phagocytic macrophages selectively in mice. After the ingestion of Clo-lip into macrophages, phospholipases in the lysosomes degrade the phospholipid bilayers, releasing the clodronate into the cells, resulting in cell death, and the i.v. injection of Clo-lip into mice results in a selective depletion of macrophages, including liver and spleen macrophages [21 , 22 ]. This study demonstrated that the three-time administration of Clo-lip eliminated F4/80+ macrophages in the liver and spleen (Fig. 1) , suggesting that the Clo-lip treatment depletes phagocytic macrophages in mice and that phagocytic macrophages are not critically involved in the increase in serum IL-18 levels. The present study also used GdCl3 to inactivate phagocytic macrophages, as it is reported that the i.v. injection of GdCl3 not only blocks phagocytosis but also eliminates these cells [24 , 25 ]; however, we were unable to deplete F4/80+ macrophages in the liver and spleen by the i.v. injection of GdCl3 three times (Fig. 1) , and the administration of GdCl3 three times showed no obvious effect on serum IL-18 levels, which were comparable with Clo-lip (Fig. 2) . It is also reported that GdCl3 does not significantly reduce the number of phagocytically active cells in the liver [31 ], that splenic macrophages are less vulnerable to GdCl3 [25 ], and that GdCl3 treatment results in a significant increase in serum levels of TNF-{alpha}, IL-6, and liver enzymes, ALT and AST [32 ]. Therefore, we consider that Clo-lip is more effective in the inactivation and elimination of phagocytic macrophages than GdCl3, and we used Clo-lip mainly.

Analysis using IFN-{gamma}-deficient mice showed that the induction of LPS hypersensitivity by P. acnes priming is mediated strictly by IFN-{gamma} [33 , 34 ]. IL-18 with IL-12 strongly induces IFN-{gamma} from activated CD4+ Th1 cells and NK cells [1 ], and IFN-{gamma} also in turn can regulate the secretion of bioactive IL-18, constituting a feedback loop between these cytokines [33 ]. IFN-{gamma} plays a major role in immune regulation. It activates various macrophage functions, including cytokine production, antimicrobial activity, and antigen processing and presentation [35 ]. In addition, IFN-{gamma} activates nonimmune cells, including keratinocytes, epithelial cells, and fibroblasts [36 37 38 ]. IFN-{gamma}-activated keratinocytes express a number of chemokines, cytokines, and adhesion molecules [36 , 37 ]. The priming of oral epithelial cells and gingival fibroblasts with IFN-{gamma} induces sensitivity to pathogen-associated molecular patterns, including LPS and peptidoglycans [38 , 39 ]. Therefore, the induction of IFN-{gamma} during P. acnes priming in mice is critically involved in the accumulation of a mature IL-18 from each organ.

The IL-18 levels were increased in the liver and spleen but decreased in the intestines by P. acnes and LPS compared with P. acnes alone (Fig. 3A) . Immunoblot analysis showed that proIL-18 was detected mainly in the liver and spleen in untreated mice (Figs. 3B and 4B) , whereas the diverse expression of proIL-18 and mature IL-18 was observed in the intestines of mice (Fig. 3B) . Furthermore, treatment with P. acnes and LPS induced a marked increase in IL-18 levels in the liver and spleen but decreased them slightly in the intestines compared with those with P. acnes alone. Therefore, it is possible that the mechanism of accumulation and release of IL-18 are different depending on the organ and even in the intestines.

F4/80 is expressed on most resident tissue macrophages, including the red pulp macrophages in the spleen, Kupffer cells in the liver, and Langerhans cells in the skin [40 ]. This study showed that the Clo-lip treatment did not reduce the IL-18 levels in the livers and spleens of mice treated with P. acnes and LPS (Fig. 4) . These findings suggest that the tissues or cells other than activated macrophages also express IL-18 in the liver and spleen. DC are able to produce IL-18 [41 ] and are not phagocytic [42 ], and the phenotypes of hepatic DC are F4/80low or F4/80– [43 ]. A recent study revealed that DC in the liver play important roles in the induction and regulation of immune responses [43 ]. Therefore, it is possible that the administration of Clo-lip did not efficiently eliminate the IL-18-expressing DC in the liver. This possibility may be the case in the spleen, as there is no report that T and B cells produce IL-18. However, murine bone marrow-derived DC produce IL-18 at ~100 pg/ml levels for 7 or 8 days of culture [41 ], whereas serum IL-18 levels in mice treated with P. acnes and LPS were ng/ml levels (Fig. 2) . Therefore, it is conceivable that the contribution of CD-derived IL-18 to serum IL-18 levels is marginal. Another possible source in the liver is oval cells, which are proliferating, epithelial cells with an ovoid nucleus and appear in liver generation, and IL-18 was expressed in oval cells in the regenerating liver at mRNA and protein levels [44 ]. Therefore, it is also possible that pathological changes caused by P. acnes induce IL-18-expressing oval cells in the liver. NK cells or NK T cells may also be the source of IL-18. Further studies are required to clarify these points.

It has been reported that treatment with P. acnes and LPS induces IL-18-dependent liver injury through the induction of TNF-{alpha} [28 29 30 ] and suggested that Fas/Fas ligand-mediated IL-18 secretion from Kupffer cells causes the liver injury in mice [29 ]. Analysis using IL-18 transgenic mice showed that IL-18 plays a key role in regulating hepatocyte apoptosis in vivo [45 ], indicating that IL-18 is critically involved in the liver injury. This study showed that depletion of F4/80+ macrophages by Clo-lip in P. acnes and LPS-treated mice reduced serum AST, ALT, and TNF-{alpha} levels and pathological change of the liver (Figs. 5 and 6) . These results suggest that Kupffer cells play an important role for the onset of liver injury. However, a substantial amount of the liver enzymes and the liver injury was found in the Clo-lip-treated mice, suggesting that IL-18 and TNF-{alpha} from liver tissues or cells other than Kupffer cells also cause the liver injury. It is also possible that IL-18 in circulation is involved in the liver injury, as the i.p. injection of rIL-18 is able to induce the liver injury in P. acnes-primed mice [29 ].

In conclusion, the present study suggests that serum IL-18 is derived from nonphagocytic macrophages, probably from epithelial cells of various organs and tissues in mice treated with P. acnes and LPS. Increased levels of serum IL-18 are associated with a wide variety of diseases [6 7 8 9 10 11 12 13 14 15 16 ]; therefore, it is possible that the serum IL-18 is derived from diseased organs or tissues. IL-18 is not only an important regulator of innate and acquired immune responses but also a potent, proinflammatory cytokine, which regulates a wide variety of autoimmune and inflammatory diseases [2 3 4 ]. Bone malformation and the exacerbation of colitis were reported in IL-18 transgenic mice [46 , 47 ], and the overexpression of IL-18 with the Keratin 5 promoter in mice showed exacerbated and prolonged, allergic and nonallergic, inflammatory skin reactions [48 ]. These findings indicate that the overexpression of IL-18 results in deleterious alterations in the organs and tissues. Therefore, IL-18 overexpressed in these organs and tissues may be an important, therapeutic target for the treatment of diseases.


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ACKNOWLEDGEMENTS
 
This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (17390483, 18591995, and 18791379) and the 21st Century Centers of Excellence Program Special Research grant from the Ministry of Education, Culture, Sports, Science and Technology, Japan. We thank T. Hoshino for providing us with anti-mouse IL-18 polyclonal antibody.

Received October 2, 2006; revised April 10, 2007; accepted April 25, 2007.


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