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Originally published online as doi:10.1189/jlb.0904510 on December 23, 2004

Published online before print December 23, 2004
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(Journal of Leukocyte Biology. 2005;77:400-407.)
© 2005 by Society for Leukocyte Biology

Differential effect of LFA703, pravastatin, and fluvastatin on production of IL-18 and expression of ICAM-1 and CD40 in human monocytes

Hideo Kohka Takahashi*,{dagger},{ddagger}, Shuji Mori*, Hiromi Iwagaki{dagger}, Tadashi Yoshino{ddagger}, Noriaki Tanaka{dagger}, Gabriele Weitz-Schmidt§ and Masahiro Nishibori*,1

* Departments of Pharmacology,
{dagger} Gastroenterological Surgery, Transplant, and Surgical Oncology, and
{ddagger} Pathology, Okayama University Graduate School of Medicine and Dentistry, Japan; and
§ Novartis Institutes for Biomedical Research, Basel, Switzerland

1 Correspondence: Department of Pharmacology, Okayama University Graduate School of Medicine and Dentistry, 2-5-1 Shikata-cho, Okayama 700-8558, Japan. E-mail: mbori{at}md.okayama-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
A novel, proinflammatory cytokine, interleukin (IL)-18 production was detected in the medium of human monocytes treated with 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase inhibitors, pravastatin, and fluvastatin (0.1 and 1 µM) but not with the statin-derived lymphocyte function-associated antigen-1 (LFA-1) inhibitor LFA703, which did not inhibit HMG-CoA reductase. Pravastatin and fluvastatin also induced the production of IL-18, tumor necrosis factor {alpha} (TNF-{alpha}) and interferon-{gamma} (IFN-{gamma}) in human peripheral blood mononuclear cells (PBMC) in contrast to LFA703. IL-18 production by PBMC is located upstream of the cytokine cascade activated by these statins. The IL-18-induced cytokine production was demonstrated to be dependent on adhesion molecule expression on monocytes. In the absence and presence of lower concentrations (0.1 and 1 ng/ml) of IL-18, pravastatin and fluvastatin inhibited the expression of intercellular adhesion molecule (ICAM)-1 and induced the expression of CD40, whereas LFA703 had no effect. In the presence of higher concentrations (5, 10, and 100 ng/ml) of IL-18, pravastatin, fluvastatin, and LFA703 similarly inhibited the expression of ICAM-1 and CD40 as well as the production of IL-12, TNF-{alpha}, and IFN-{gamma} in PBMC. The effects of pravastatin and fluvastatin but not LFA703 were abolished by the addition of mevalonate, indicating the involvement of HMG-CoA reductase in the action of pravastatin and fluvastatin. Thus, the effects of LFA703 were distinct from those of pravastatin and fluvastatin in the presence of lower concentrations of IL-18. It was concluded that LFA703 has the inhibitory effect on an IL-18-initiated immune response without any activation on monocytes.

Key Words: human • peripheral blood mononuclear cells • 3-hydroxy-3-methylglutaryl coenzyme-A


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Interleukin (IL)-18, a monocyte-derived cytokine, requires the cleavage at its aspartic acid residue by IL-1ß-converting enzyme/caspase-1 to become the active, mature protein [1 , 2 ]. IL-18 stands up-stream of the production of T helper cell type 1 (Th1) cytokines [3 4 5 ]. Cell-to-cell interactions mediated by the engagement of adhesion molecules [intercellular adhesion molecule-1 (ICAM-1), B7.1/B7.2, and CD40] on monocytes and their ligands [lymphocyte function-associated antigen-1 (LFA-1), CD28, and CD40L] on T/natural killer cells are involved in the production of IL-18-initiated cytokines including IL-12, tumor necrosis factor {alpha} (TNF-{alpha}), and interferon-{gamma} (IFN-{gamma}) in acquired [6 ] and innate immune responses [7 , 8 ]. IL-18 has been suggested to be involved in the pathogenesis of diseases such as rheumatoid arthritis (RA), ischemia/reperfusion injury, and transplant rejection [9 10 11 ].

Statins reduce total low-density lipoprotein cholesterol via inhibiting 3-hydroxy-3-methylglutaryl coenzyme-A (HMG-CoA) reductase, a rate-limiting enzyme for cholesterol biosynthesis [12 ]. Recent studies have demonstrated that statins have modulatory effects on inflammatory and immune responses [13 14 15 16 17 18 19 ]. Treatment with fluvastatin, an inhibitor of HMG-CoA reductase, resulted in the activation of caspase-1 and secretion of some amount of IL-1ß, IL-18, and IFN-{gamma} in peripheral blood mononuclear cells (PBMC) [16 ]. It is proposed that statins may have host-defense effects against infections by enhancing the Th1 response [16 ]. Conversely, the treatment of human-cultured endothelial cells and monocytes with pravastatin (0–10 µM) inhibited CD40 expression stimulated by TNF-{alpha} plus IFN-{gamma} [17 ]. By interfering with cytokine-stimulated CD40 expression in vascular endothelial cells, statins are thought to attenuate CD40L-induced proinflammatory responses including the development of atherosclerosis [14 ]. Thus, it is implied that statins may have dual inflammatory and anti-inflammatory effects. The ß2 integrin LFA-1 has an important role in the inflammatory and autoimmune diseases. LFA703, a structural analog of statin compounds but without an inhibitory effect on HMG-CoA reductase [19 ], selectively blocked LFA-1-mediated adhesion and costimulation of lymphocytes through the binding to a novel allosteric site within LFA-1 [18 ], whereas pravastatin did not bind to LFA-1 [18 ]. Targeting the statin-binding site of LFA-1 could treat diseases [18 ], in which IL-18 production may exert the specific roles in pathogenesis [9 10 11 ].

In the present study, we examined the effect of fluvastatin, pravastatin, and LFA-703 on the production of IL-18 in human monocytes and the IL-18-elicited expression of ICAM-1, B7.1, B7.2, CD40, and CD40L.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and drugs
Recombinant human IL-18, anti-IL-18 monoclonal antibodies (mAb), and caspase-1 inhibitor Z-tyr-Val-Ala-Asp-fluoromethyl ketone (YVAD-FMK) were purchased from MBL (Nagoya, Japan). Novartis (Basel, Switzerland) kindly provided LFA703 and fluvastatin. Sankyo (Tokyo Japan) kindly provided pravastatin. Mevalonate was purchased from Sigma Aldrich (St. Louis, MO). For flow cytometric analysis, fluorescein isothoicyanate (FITC)-conjugated mouse immunoglobulin G1 (IgG1) mAb against ICAM-1 and phycoerythrin-conjugated anti-CD14 mAb were purchased from Dako (Glostrup, Denmark). FITC-conjugated mouse IgG1 mAb against B7.1 were purchased from Immunotech (Marseille, France). FITC-conjugated mouse IgG1 mAb against B7.2 and CD40 were purchased from PharMingen (San Diego, CA). FITC-conjugated mouse IgG1 mAb against CD40L were purchased from Ancel (Bayport, MN). FITC-conjugated IgG1 class-matched control (CMC) was purchased from Sigma Aldrich.

Preparation of isolated monocyte
Normal human PBMC were obtained from 10 human volunteers with their oral, informed consent. Samples of 50 ml peripheral blood were withdrawn from a forearm vein. PBMC were prepared as described previously [7 , 8 ]. Separation of monocytes from PBMC was conducted by counterflow centrifugal elutriation as described previously [8 ]. Monocytes were suspended at a final concentration of 1 x 106 cells/ml in RPMI-1640 medium (Nissui Co. Ltd., Tokyo, Japan) supplemented with 10% (v/v) heat-inactivated fetal calf serum, 20 µg/ ml kanamycin, and 100 µg/ ml streptomycin and penicillin (Sigma Aldrich). The cell population of monocytes was 85%, determined by flow cytometry with FITC-conjugated anti-CD14 antibody.

Flow cytometric analysis
Monocytes (1x106 cells/ ml) were incubated with LFA703, pravastatin, fluvastain, anti-IL-18 mAb, and/or YVAD-FMK in the presence and absence of IL-18 for 24 h at 37°C in a 5% CO2/air mixture under different conditions. The cells (5x105 cells/ sample) were stained with 1 µg FITC-conjugated anti-ICAM-1, anti-B7.1, anti-B7.2, anti-CD40, and anti-CD40L antibodies or CMC for 20 min at 4°C as described previously [7 , 8 ]. After washing, the cells were fixed with 2% paraformaldehyde. The adhesion molecule expression was analyzed on 10,000 cell monocytes for each sample with a FACSCalibur (BD Biosciences, San Jose, CA), and data were processed using the CellQuest program (BD Biosciences). The data are expressed as the relative fluorescence intensities against CMC. The results are the means ± SEM of five donors.

Cytokine assay
The cell-free supernatant fractions of monocytes and PBMC incubated as described in flow cytometric analysis were assayed for IL-18, IL-12 (p70), TNF-{alpha}, IFN-{gamma}, and IL-10 protein by enzyme-linked immunosorbent assay (ELISA) using commercially available kits (IL-18, MBL; IL-12, TNF-{alpha}, IFN-{gamma}, IL-10, quantikine, R&D Systems, Minneapolis, MN) as described previously [7 , 8 ].

Statistical examination
The statistical significance of differences was evaluated by ANOVA followed by Dunnet’s test. A probability value less than 0.05 was considered significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of LFA703, fluvastatin, and pravastatin on the production of IL-18 in monocytes
Monocytes (1x106 cells/ml) were treated with LFA703, fluvastatin, and pravastatin (0–1 µM). After 24 h incubation, the production of IL-18 was determined by ELISA. Pravastatin and fluvastatin concentration-dependently induced the production of IL-18, whereas LFA703 had no effect (Fig. 1 ). The level of IL-18 production stimulated by pravastatin and fluvastatin (1 µM) reached ~400 pg/ml. Conversely, LFA703, pravastatin, and fluvastatin had no effect on the production of IL-12, TNF-{alpha}, IFN-{gamma}, and IL-10 in monocytes in the presence and absence of IL-18 (data not shown).



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  		Figure 1. The effect of LFA703, fluvastatin, and pravastatin on the production of IL-18 in monocytes. Monocytes (1x106 cells/ml) were incubated with increasing concentrations (0–1 µM) of LFA703, fluvastatin, and pravastatin for 24 h, and the level of IL-18 in the conditioned media was determined by ELISA. ({circ}, •, {blacksquare}) Results obtained with LFA703, fluvastatin, and pravastatin, respectively. **, P < 0.01, compared with the corresponding value in medium alone. The results are the means ± SEM of five donors. When an error bar was within a symbol, the bar was omitted.

 
The kinetics of the effect of LFA703, fluvastatin, and pravastatin on the production of IL-18, IL-12, TNF-{alpha}, IFN-{gamma}, and IL-10 in PBMC
The time-course changes in cytokine production in PBMC induced by LFA703, fluvastatin, and pravastatin (1 µM) in the presence and absence of IL-18 (100 ng/ml) were examined 0, 4, 16, and 24 h after the start of incubation. In the absence of IL-18, pravastatin and fluvastatin, but not LFA703, induced the production of IL-18, TNF-{alpha}, and IFN-{gamma} (without detectable changes in IL-12 production; data not shown) and inhibited IL-10 production (Fig. 2A ). The effects of pravastatin and fluvastatin on cytokine production were significant at 16 and 24 h. Conversely, LFA703, fluvastatin, and pravastatin inhibited the IL-18-enhanced production of IL-12, TNF-{alpha}, and IFN-{gamma} but reversed the IL-18-suppressed production of IL-10 at 16 and 24 h, respectively (Fig. 2B) .



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  		Figure 2. The kinetics of the effect of LFA703, fluvastatin, and pravastatin on the production of IL-18, IL-12, TNF-{alpha}, IFN-{gamma}, and IL-10 in PBMC. (A) PBMC (1x106 cells/ml) were incubated with LFA703, fluvastatin, and pravastatin (1 µM) for 0, 4, 16, or 24 h. IL-18, TNF-{alpha}, IFN-{gamma}, and IL-10 production was determined by ELISA. **, P < 0.01, compared with the value in the presence of medium alone. (x, {circ}, •, {blacksquare}) Results obtained with medium, LFA703, fluvastatin, and pravastatin, respectively. (B) PBMC (1x106 cells/ml) were incubated with LFA703, fluvastatin, and pravastatin (1 µM) in the presence of IL-18 (100 ng/ml) for 0, 4, 16, or 24 h. IL-12, TNF-{alpha}, IFN-{gamma}, and IL-10 production was determined by ELISA. (x, {circ}, •, {blacksquare}) Results obtained with IL-18, LFA703, fluvastatin, and pravastatin, respectively. ##, P < 0.01, compared with the value in the presence of IL-18 alone. The results are the means ± SEM of five donors. When an error bar was within a symbol, the bar was omitted.

 
Dose-response relationships of the effect of LFA703, fluvastatin, and pravastatin on the production of IL-18, IL-12, TNF-{alpha}, IFN-{gamma}, and IL-10 in PBMC
PBMC (1x106 cells/ml) were treated with different concentrations (0–1 µM) of LFA703, fluvastatin, and pravastatin in the presence and absence of IL-18 (100 ng/ml). In the absence of IL-18, pravastatin and fluvastatin induced IL-18, TNF-{alpha}, and IFN-{gamma} production (without detectable changes in IL-12 production; data not shown) but inhibited IL-10 production (Fig. 3A ). In contrast, LFA703 had no effect. In the presence of IL-18, pravastatin, fluvastatin, and LFA703 inhibited the production of IL-12, TNF-{alpha}, and IFN-{gamma} but induced the production of IL-10 with similar potencies (Fig. 3B) .



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  		Figure 3. The dose-response relationships of the effect of LFA703, fluvastatin, and pravastatin on the production of IL-18, IL-12, TNF-{alpha}, IFN-{gamma}, and IL-10 in PBMC. (A) PBMC (1x106 cells/ml) were incubated with LFA703, fluvastatin, and pravastatin (0–1 µM) for 24 h. IL-18, TNF-{alpha}, IFN-{gamma}, and IL-10 production was determined by ELISA. **, P < 0.01, compared with the value in medium alone. (B) PBMC (1x106 cells/ml) were incubated with LFA703, fluvastatin, and pravastatin (0–1 µM) in the presence of IL-18 (100 ng/ml) for 24 h. IL-12, TNF-{alpha}, IFN-{gamma}, and IL-10 production was determined by ELISA. ({circ}, •, {blacksquare}) Results obtained with LFA703, fluvastatin, and pravastatin, respectively. **, P < 0.01, compared with the value in the presence of IL-18 alone. The results are the means ± SEM of five donors. When an error bar was within a symbol, the bar was omitted. M, molar.

 
Effect of LFA703, fluvastatin, and pravastatin on the expression of ICAM-1, B7.1, B7.2, CD40, and CD40L on monocytes
Monocytes (1x106 cells/ml) were treated with LFA703, fluvastatin, and pravastatin (0–1 µM) in the presence of IL-18 (0–100 ng/ml). After 24 h incubation, the expression of ICAM-1 and CD40 as well as the expression of B7.1, B7.2, and CD40L were determined by flow cytometry (Fig. 4 ). At 0.1 and 1 ng/ml, IL-18 had a weak effect. LFA703 had no effect on the expression of ICAM-1 and CD40 under these conditions, whereas pravastatin and fluvastatin inhibited the expression of ICAM-1 and induced the expression of CD40 in a concentration-dependent manner. At 5, 10, and 100 ng/ml, IL-18 up-regulated the expression of ICAM-1, B7.2, and CD40 but had no effect on B7.1 and CD40L expression. Under these ranges of IL-18 concentrations, LFA703, fluvastatin, and pravastatin inhibited the expression of ICAM-1 and CD40. Inhibitory concentration 50% values for the inhibitory effect of LFA703, fluvastatin, and pravastatin on the expression of ICAM-1 in the presence of IL-18 (100 ng/ml) were estimated to be 200, 300, and 300 nM. The expression of B7.1, B7.2, and CD40L was not changed by these statins in the presence and absence of IL-18 (data not shown).



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  		Figure 4. The effect of LFA703, fluvastatin, and pravastatin on the expression of ICAM-1 and CD40 on monocytes. Monocytes (1x106 cells/ml) were incubated with IL-18 (0–100 ng/ml), LFA703 (A), fluvastatin (B), and pravastatin (C; 0–1 µM) for 24 h. After the treatment, the expression of ICAM-1 and CD40 was determined by flow cytometry. (+) Relative fluorescence intensities against class-matched control. (x, {circ}, {square}, •, {blacksquare}, {diamondsuit}) Results obtained with 0, 0.1, 1, 5, 10, and 100 ng/ml IL-18, respectively. The results are the means ± SEM of five donors. *, P < 0.05; **, P < 0.01, as compared with the corresponding value in the absence of LFA703, fluvastatin, or pravastatin. When an error bar was within a symbol, the bar was omitted. M, molar.

 
Effects of anti-IL-18 mAb and YVAD-FMK on cytokine responses induced by pravastatin and fluvastatin in PBMC
PBMC (1x106 cells/ml) were treated with anti-IL-18 mAb (10 ng/ml) and caspase-1 inhibitor (YVAD-FMK; 100 nM) in the presence of pravastatin or fluvastatin (1 µM; Table 1 ). After 24 h incubation, the production of IL-18, IL-12, TNF-{alpha}, IFN-{gamma}, and IL-10 was determined by ELISA. IL-18 (1 ng/ml) induced the production of IFN-{gamma} (200 pg/ml), TNF-{alpha} (150 pg/ml), and IL-12 (22 pg/ml), whereas the same concentration of IL-18 inhibited the production of IL-10 (370 pg/ml). Pravastatin and fluvastatin (1 µM) induced the production of IL-18 (400 pg/ml), IFN-{gamma} (150 pg/ml), and TNF-{alpha} (150 pg/ml) without IL-12 production but inhibited the production of IL-10 (370 pg/ml). Anti-IL-18 mAb abolished the effect of 1 ng/ml IL-18 on the cytokine production. The treatment of anti-IL-18 mAb and YVAD-FMK inhibited the enhanced production of TNF-{alpha} and IFN-{gamma} induced by pravastatin and fluvastatin but induced the production of IL-10.


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  		Table 1. Effects of Anti-IL-18 mAb and Caspase-1 Inhibitor (YVAD-FMK) on Cytokine Responses Induced by Pravastatin and Fluvastatin in PBMC

 
Effect of mevalonate on the statin-initiated production of IL-18, IL-12, TNF-{alpha}, IFN-{gamma}, and IL-10 in monocytes and PBMC
Monocytes (1x106 cells/ml) were treated with mevalonate (0–100 µM) in the presence of LFA703, fluvastatin, or pravastatin (1 µM). After 24 h incubation, the production of IL-18 was determined by ELISA. In the absence of LFA703, fluvastatin, or pravastatin, mevalonate alone had no effect on cytokine production (data not shown). Mevalonate abolished pravastatin- or fluvastatin-enhanced IL-18 production in monocytes (Fig. 5A ). LFA703 remained inactive in this assay in the presence of mevalonate in monocytes and PBMC (Fig. 5) . Mevalonate suppressed pravastatin- or fluvastatin-induced TNF-{alpha} and IFN-{gamma} production in PBMC but enhanced IL-10 production (Fig. 5B) . Moreover, mevalonate reversed the modulating effect of pravastatin or fluvastatin (1 µM) on IL-18-initiated production of TNF-{alpha}, IFN-{gamma}, and IL-10 in PBMC (Fig. 5C) . In contrast, mevalonate did not reverse the inhibitory effect of LFA703 on cytokine production in PBMC (Fig. 5)



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  		Figure 5. The effect of mevalonate on the modulatory effect of LFA-703, fluvastatin, and pravastatin on the production of IL-18, IL-12, TNF-{alpha}, IFN-{gamma}, and IL-10 in monocytes and PBMC. (A) Monocytes (1x106 cells/ml) were treated with mevalonate (0–100 µM) in the presence of LFA703, fluvastatin, or pravastatin (0–1 µM), and the level of IL-18 in the conditioned media was determined by ELISA. *, P < 0.05; ** P < 0.01, compared with the corresponding value in the presence of LFA703, fluvastatin, or pravastatin alone (1 µM). (B) PBMC (1x106 cells/ml) were incubated with with mevalonate (0–100 µM) in the presence of LFA703, fluvastatin, or pravastatin (1 µM) for 24 h. TNF-{alpha}, IFN-{gamma}, and IL-10 production was determined by ELISA. *, P< 0.05; **, P < 0.01, compared with the corresponding value in the presence of LFA703, fluvastatin, or pravastatin alone. (C) PBMC (1x106 cells/ml) were treated with mevalonate (0–100 µM) in the presence of LFA703, fluvastatin, or pravastatin (1 µM) with IL-18 (100 ng/ml) for 24 h. IL-12, TNF-{alpha}, IFN-{gamma}, and IL-10 production was determined by ELISA. *, P < 0.05; **, P < 0.01, compared with the corresponding value in the presence of LFA703, fluvastatin, or pravastatin alone. ({circ}, •, {blacksquare}) Results obtained with LFA703, fluvastatin, and pravastatin, respectively. The results are the means ± SEM of five donors. When an error bar was within a symbol, the bar was omitted. log (M), log (Molar).

 
Effect of mevalonate on the action of statin on the expression of ICAM-1 and CD40L on monocytes
Monocytes (1x106 cells/ml) were treated with mevalonate (0–100 µM) in the presence of LFA703, fluvastatin, or pravastatin(1 µM). After 24 h incubation, the expression of ICAM-1 and CD40 was determined by flow cytometry. Mevalonate had no effect on the expression of ICAM-1 and CD40 (data not shown). Mevalonate concentration-dependently reversed the modulatory effects of pravastatin or fluvastatin on the expression of ICAM-1 and CD40 in the absence of IL-18 (Fig. 6A ). Moreover, mevalonate antagonized the modulatory effect of pravastatin or fluvastatin on the expression of ICAM-1 and CD40 in the presence of high concentration of IL-18 (100 ng/ml; Fig. 6B ). In the presence of LFA703, mevalonate had no effect, irrespective of the presence of IL-18 (Fig. 6) .



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  		Figure 6. The effect of mevalonate on the modulatory effect of LFA-703, fluvastatin, and pravastatin on the expression of ICAM-1 and CD40L on monocytes (1x106 cells/ml), which were incubated with mevalonate (0–100 µM) in the presence of LFA703, fluvastatin, and pravastatin (1 µM) with (A) or without (B) IL-18 (100 ng/ml) for 24 h. After the treatment, the expression of ICAM-1 and CD40 was determined by flow cytometry. ({square}, {circ}, {lozenge}) Results obtained with LFA703, fluvastatin, and pravastatin, respectively. ({blacksquare}, •, {diamondsuit}) Results obtained with LFA703, fluvastatin, and pravastatin in the presence of IL-18, respectively. The results are the means ± SEM of five donors. *, P < 0.05; **, P < 0.01, as compared with the corresponding value in the presence of LFA703, fluvastatin, or pravastatin alone. When an error bar was within a symbol, the bar was omitted. log (M), log (Molar).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The effect of LFA703 on IL-18 production was different from those of pravastatin and fluvastatin. Consistent with the result that fluvastatin (5 µM) induced IL-18 production (200 pg/ml) via caspase-1 activation in PBMC [16 ], pravastatin and fluvastatin (1 µM) but not LFA703 induced IL-18 production (400 pg/ml) in monocytes (Fig. 1) . The result that LFA703, pravastatin, and fluvastatin had no effect on IL-12, TNF-{alpha}, IFN-{gamma}, and IL-10 production in monocytes (data not shown) was consistent with the previous study showing that IL-18 has no effect on the production of IL-12, TNF-{alpha}, IFN-{gamma}, and IL-10 in isolated monocytes [20 ]. IL-18-initiated cytokine production requires cell-to-cell interaction between monocytes and T cells [7 , 8 ]. As the amount of IL-12 production was level low in the medium of PBMC treated with 1 ng/ml IL-18 (Table 1 and ref. [21 ]), 400 pg/ml IL-18 induced by pravastatin and fluvastatin resulted in the lack of IL-12 production (Fig. 3A) . As shown in Table 1 , IL-18 production in PBMC is located up-stream of the cytokine cascade activated by pravastatin and fluvastatin.

Whereas pravastatin and fluvastatin mimicked the effect of histamine, prostaglandin, and epinephrine on IL-18 production [7 , 20 , 21 ], the effect of pravastatin and fluvastatin on adhesion molecule expression was distinct from that of histamine, prostaglandin, and epinephrine in the absence of IL-18. As shown in Figure 4 B and C , pravastatin and fluvastatin inhibited ICAM-1 expression and induced CD40 expression. Although we demonstrated that IL-18 (1 ng/ml) up-regulated the expression of CD40 on monocytes in the previous study [8 ], the addition of anti-IL-18 mAb (10 ng/ml) and YVAD-FMK (100 nM), which abolished the effect of 1 ng/ml IL-18 on the expression of CD40, did not change the effect of pravastatin or fluvastatin alone (1 µM) on the expression of CD40 on monocytes (data not shown). Therefore, it is unlikely that endogenously produced IL-18 by pravastatin or fluvastatin was a mediator for the elevation of CD40 expression on monocytes. Although IL-18 induces ICAM-1 expression on monocytes [7 ], pravastatin or fluvastatin suppressed ICAM-1 expression (Fig. 4B and 4C) . It was reported that fluvastatin at 0.1 µM or more inhibited the expression of ICAM-1 on a monocytic cell line U937 [22 ]. Thus, pravastatin and fluvastatin appeared to have additional effects on IL-18 production in monocytes.

Conversely, the effect of LFA703, pravastatin, and fluvastatin on cytokine production and adhesion molecule expression was similar in the presence of IL-18 (Figs. 3B and 4) . Anti-ICAM-1 and anti-B7.2 antibodies abolished IL-18-initiated IL-12, TNF-{alpha}, IFN-{gamma}, and IL-10 production in PBMC [7 ], whereas anti-CD40 antibody had no effect on IL-18-modulated cytokine production (data not shown). Therefore, the effects of statins on cytokine production in the presence of IL-18 suggest that adhesive events mediated by the LFA-1/ICAM-1 pathway are involved in the action of statins. Cell viability of monocytes and lymphocytes in the presence of LFA703, pravastatin, fluvastatin, and/or IL-18 was almost the same and was estimated to be 85–90% after 24 h incubation. These results suggested that the regulation of cytokine production and adhesion molecule expression was not a result of reduction in cell viability. Simvastatin, lovastatin, pravastatin, or atorvastatin was reported to reduce anti-CD3/anti-CD28-induced proliferation of T cells and IFN-{gamma} release in PBMC [13 ] and to decrease IFN-{gamma}-induced CD40 and ICAM-1 expression on vascular endothelial and smooth muscle cells [14 , 15 ]. Thus, statins might have anti-inflammatory effects through the action on plural cell types, monocytes, T cells, and vascular endothelial cells. A recent study reported that the effect of LFA703 depends on the binding to a novel allosteric site within LFA-1 [18 ] but not on the inhibitory effect on HMG-CoA reductase [19 ]. The product of the HMG-CoA reductase, mevalonate abolished all modulatory effects of pravastatin and fluvastatin on cytokine production and adhesion molecule expression but had no effect on the LFA-703 action (Figs. 5 and 6) . Therefore, it was concluded that the effects of pravastatin and fluvastatin but not LFA-703 on cytokine production and adhesion molecule expression were mediated by the inhibition of HMG-CoA reductase. The lack of an effect of mevalonate on the LFA-703 action suggests that the compound exhibits the described effects on cytokine production via direct inhibition of the LFA-1/ICAM-1 interaction. LFA-1 is also known to be expressed on monocytes. The blocking of LFA-1/ICAM-1 engagement between monocytes might contribute to the inhibitory effect of LFA-703 on IL-18-enhanced adhesion molecule expression. Statin therapy is suggested to have beneficial effects on allergic asthma [23 ], organ rejection after transplantation [14 , 20 ], and RA [10 ], in which IL-18 has important roles in their pathogenesis [9 10 11 ]. It was reported that elevated levels of serum IL-18 were consistent with a pathophysiological role of IL-18 in RA [10 ]. Therefore, the clinical effects of these statins on asthma, organ rejection after transplantation, and RA might depend on the inhibition of IL-18-induced adhesion molecules expression.

By using a mouse model, LFA703 was shown to exert anti-inflammatory effects through regulation of LFA-1-mediated adhesion and costimulation of lymphocytes [18 , 19 , 24 ]. The LFA703-induced interference with LFA-1-mediated leukocyte-endothelium interactions, which are associated with leukocyte activation and recruitment in the liver, protects against endotoxemic liver damage and constitutes a potential therapeutic strategy in sepsis [24 ]. Immunoneutralization of LFA-1 significantly reduced ischemia/reperfusion leukocyte adhesion in colonic venules [19 ]. Thus, LFA703 is useful in the clinical treatment of conditions associated with ischemia/reperfusion-induced tissue injury, such as organ transplantation and major surgery. The lack of the effect of LFA-703 alone in producing cytokines may have therapeutic potentials for certain stages of inflammatory and autoimmune diseases. Further studies on the effects of statins on immune response should be continued.


   ACKNOWLEDGEMENTS
 
This study was supported in part by a grant for Promotion of Research from Okayama University (No. 21 to M. N.), a grant from Okayama Medical Foundation (to H. K. T.), and grants from Grant-in-Aid for Scientific Resarch (15590467 to H. K. T. and 15590228 to M. N.). Novartis kindly provided LFA703 and fluvastatin. Sankyo kindly provided pravastatin. The authors thank Ms. Yumiko Shiotani and Ms. Yuki Onoda for excellent technical assistance.

Received September 11, 2004; revised November 18, 2004; accepted November 19, 2004.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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