Introduction

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It has been widely accepted that cytokines such as tumor necrosis factor- and interleukin-1, and also arachidonic acid metabolites, have an important role in inflammatory diseases such as rheumatoid arthritis, systemic lupus erythematosus, asthma, inflammatory bowel disease, psoriasis, and other chronic inflammatory and autoimmune diseases. Glucocorticoids are potent anti-inflammatory agents widely used in those inflammatory conditions. Glucocorticoids exert their effects by binding to a cytoplasmic glucocorticoid receptor within the target cells. The glucocorticoid-receptor complex then translocates to the nucleus and modulates the expression of specific target genes in a positive or negative manner, interfering with multiple signaling pathways. Consequently, a wide variety of genes are regulated by glucocorticoids (Barnes, 1998; McKay and Cidlowski, 1999). Therefore, although glucocorticoids are effective for treatment of inflammatory diseases, their use is limited by their severe adverse effects, such as increased susceptibility to infection, osteoporosis, delayed wound healing, and so on (Stein and Pincus, 1997).

In recent years, pyridinyl imidazole derivatives have been reported as a novel class of cytokine synthesis inhibitors (Lee et al., 1993, 1994). These agents inhibit the production of specific cytokines including tumor necrosis factor-, interleukin-1, interleukin-6, and interleukin-8 in an in vitro system (Lee et al., 1993). It is reported that the target of these inhibitors is a pair of closely related protein kinases, which are human homologs of p38 mitogen-activated protein kinase, termed cytokine-suppressive anti-inflammatory drug-binding protein (CSBP). The binding of these drugs inhibits CSBP kinase activity and could be directly correlated with their ability to inhibit cytokine production (Lee et al., 1994). FR167653 is a pyridinyl isoimidazole derivative and is shown to inhibit cytokine production (Yamamoto et al., 1996). It inhibits the expression of tumor necrosis factor-, interleukin-1, and cyclooxygenase-2 in human blood monocytes and alveolar macrophages (Kawano et al., 1999) and also can exert protective effects on lipopolysaccharide-induced disseminated intravascular coagulation (Yamamoto et al., 1996) and endotoxin-induced shock (Yamamoto et al., 1997) in animal models.

Rat carrageenin-induced pleurisy is the most widely accepted model suitable for collection and analysis of cells and exudate from the inflammatory site. Although, carrageenin pleurisy is a neutrophil-dominated model, a number of reports have demonstrated that neutrophils also have the ability to synthesize and release cytokines (Cassatella, 1995). Using this model, we have previously shown that a high level of cyclooxygenase-2 is detectable from 3 to 7 h in neutrophils and mononuclear leukocytes (Harada et al., 1994, 1996), and from 9 to 24 h in pleural mesothelial cells (Hatanaka et al., 1999). In the early phase, selective cyclooxygenase-2 inhibitors suppress plasma exudation and preferentially reduce the prostaglandin E₂ level, but not the level of 6-keto-prostaglandin F₁ or of thromboxane B₂ in the pleural exudate, suggesting that prostaglandin E₂ generated via cyclooxygenase-2 by leukocytes in the exudate may play an important role in plasma exudation. In addition, a lower dose of dexamethasone (0.3 mg/kg) does not affect the cyclooxygenase-2 level, even showing potent anti-inflammatory activity (Kawamura et al., 2000). In the late phase, selective cyclooxygenase-2 inhibitors lower the intrapleural level of 6-keto-prostaglandin F₁, a stable metabolite of prostaglandin I₂, and inhibit hyperplasia of the pleural matrix, suggesting that cyclooxygenase-2 expressed in mesothelial cells may play a role in the synthesis of extracellular matrix through formation of prostaglandin I₂ (Hatanaka et al., 1999).

In the present study, we evaluated the effects of FR167653 on the early and late phases of rat carrageenin-induced pleurisy and on mediator-induced plasma exudation, comparing them with those of dexamethasone, to characterize its pharmacological profile.

	Materials and Methods

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Carrageenin Pleurisy. All experiments were performed according to the Guideline for Animal Experimentation of Kitasato University. Pleurisy was induced in male Sprague-Dawley rats (9-10-weeks old, specific pathogen free), purchased from Nippon SLC (Hamamatsu, Japan), by intrapleural injection of 0.2 ml of 2% -carrageenin (Zushi Chemical, Zushi, Japan) under light ether anesthesia according to the method described previously (Harada et al., 1996). For early phase experiments, FR167653 (3, 10, 30 mg/kg; Fujisawa Pharmaceutical Co., Ltd., Osaka, Japan) suspended with 1% carboxymethyl cellulose in saline had been administered orally 1 h before, and dexamethasone (0.3 mg/kg; Banyu Pharmaceutical Co., Ltd., Tokyo, Japan) had been intraperitoneally injected 2 h before the injection of carrageenin. For late phase experiments, FR167653 (30 mg/kg, p.o.) and dexamethasone (0.3 mg/kg, i.p.) were administered 9 h after the induction of pleurisy, unless otherwise stated.

Prostanoid Assay. The prostanoid level was determined as described previously (Harada et al., 1996). Briefly, the frozen pleural fluid was thawed and centrifuged at 2000g for 10 min. The supernatant was acidified to pH 3 with 1 N HCl and again centrifuged at 2000g for 10 min. The resulting supernatant was applied to a Sep-Pak C₁₈ column (Waters Associates, Milford, MA). After the separation of prostaglandin E₂, thromboxane B₂, and 6-keto-prostaglandin F₁ by high-performance liquid chromatography, prostanoid was assayed by enzyme-immunoassay kits (Cayman Chemical, Ann Arbor, MI). The overall recovery rates assessed by addition of authentic prostanoid to the sample were 52.8 ± 2.2% (n = 13), 48.9 ± 1.8% (n = 13), and 43.0 ± 2.2% (n = 13) for prostaglandin E₂, thromboxane B2, and 6-keto-prostaglandin F₁, respectively. The detection limit for each prostanoid was approximately 0.1 to 0.06 ng/sample.

Measurement of Tumor Necrosis Factor- and Interleukin-1. The frozen cell-free supernatant of the exudate was thawed, and tumor necrosis factor- and interleukin-1 were determined by enzyme-immunoassay kits (BioSource, Camarillo, CA).

Western Blot Analysis. Western blot analysis for cyclooxygenase-1 and cyclooxygenase-2 was performed by the method described previously (Harada et al., 1994). In brief, the frozen cells collected from the exudate and parietal pleura were thawed and suspended in 20 mM Tris-HCl buffer (pH 7.4), containing 5 mM tryptophan and 2 mM phenylmethyl sulfonyl fluoride (Wako Pure Chemicals), and then sonicated for 1 min at 4°C. The homogenate was then solubilized in 0.5% Tween 20 and centrifuged at 140,000g for 1 h at 4°C. The resulting supernatant was diluted with an equal volume of a sampling buffer of the following composition: 0.1 M Tris-HCl (pH 6.8), 20% glycerol, 0.1 mg/ml methyl green, and 2% sodium dodecyl sulfate. The solubilized protein (40-50 µg of protein/lane) was subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to a polyvinylidene difluoride membrane (Millipore Corporation, Bedford, MA). After blocking with Block Ace (Dainippon Pharmaceutical Co., Osaka, Japan), the blot membrane was incubated with rabbit antibovine cyclooxygenase-1 antiserum (Ishimura et al., 1993) or rabbit antimurine cyclooxygenase-2 antiserum (Cayman Chemical). Then, after incubation with goat antirabbit immunoglobulin conjugated with horseradish peroxidase (Organon Teknika N.V.-Cappel Products, Durham, NC), the membrane was stained with Konica immunostain HRP-1000 (Konica, Tokyo, Japan).

Cyclooxygenase-2 mRNA Measurement. The reverse transcription and competitive polymerase chain reaction (PCR) was used to measure cyclooxygenase-2 mRNA. The total RNA of the exudate cell pellet (approximately 4 × 10⁷ cells) from a 5-h pleurisy rat was extracted in Isogen (Nippon Gene, Tokyo, Japan), a mixture of guanidinium isothiocyanate and phenol (Chomczynski and Sacchi, 1987). The yield of RNA extracted was determined spectrophotometrically. Two micrograms of each sample was reverse-transcribed at 42°C for 1 h in buffer (50 mM Tris-HCl, pH 8.3, 75 mM KCl, 8 mM MgCl₂, and 10 mM dithiothreitol) containing 2.5 µM random hexamer oligonucleosides (TaKaRa Biomedicals, Shiga, Japan), 10 units of reverse transcriptase (RAV-II; TaKaRa Biomedicals), each of the 2'-deoxynucleoside 5'-triphosphates at 1 mM and 20 units of RNase inhibitor (TaKaRa Biomedicals).

Mediator-Induced Plasma Exudation. Male Sprague-Dawley rats (9-10-weeks old, specific pathogen-free; Nippon SLC) were anesthetized with pentobarbital (50 mg/kg, s.c.; Abbott Lab., North Chicago, IL) and were intravenously injected with pontamine sky blue (50 mg/kg; Tokyo Kasei). Intradermal injection of various doses of histamine or bradykinin in 0.1 ml of Tyrode's solution were started 5 min after injection of the dye into the shaved abdominal skin at 8 to 10 sites for each rat. The rats were sacrificed by exsanguination 40 min after the end of the injection of mediators. The exuded dye at each site was extracted by the method of Katayama et al. (1978), and its amounts were measured by spectrophotometry. FR167653 (30 mg/kg, p.o.) or dexamethasone (0.3 mg/kg, i.p.) was administered 30 min before injection of the mediators.

Data Analysis. Results were expressed as the mean ± S.E.M. from n experiments. Fisher's or Scheffe's test was used to evaluate significant differences between means. A P value of less than 0.05 was considered statistically significant and is indicated by an asterisk in the figures.

	Results

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Effects in the Early Phase of Pleurisy. The amount of pleural fluid collected from normal control rats was 0.04 ± 0.01 ml (n = 6) and that of pontamine sky blue that leaked into the pleural cavity after intravenous injection over 20 min was 2.89 ± 0.32 µg (n = 7). The total count of leukocytes harvested from the lavage fluid from the pleural cavity of normal control rats was 11.40 ± 0.94 × 10⁶ cells (n = 12). Mononuclear leukocytes accounted for 72.6 ± 2.6%. The remaining cells included eosinophils (12.8 ± 1.1%), mast cells (8.0 ± 0.9%), and neutrophils (3.5 ± 0.7%). These parameters were not significantly affected by pretreatment with FR167653 or dexamethasone (data not shown). Intrapleural injection of carrageenin caused the accumulation of a volume of pleural exudate that reached 1.57 ± 0.12 ml (n = 11), and the plasma exudation rate, estimated from the exuded dye amount, reached 167 ± 13 µg (n = 11) 5 h after the injection. The total leukocyte count was 157 ± 15 × 10⁶ (n = 6), with 91.4 ± 1.3% of neutrophils and 6.0 ± 0.5% of mononuclear leukocytes, 5 h after pleurisy induction. FR167653 (3, 10, 30 mg/kg) dose dependently suppressed both the accumulation of pleural exudate and the plasma exudation rate 5 h after carrageenin injection, as did dexamethasone (0.3 mg/kg) (Fig. 1). Furthermore, FR167653 (30 mg/kg) significantly decreased the number of neutrophils, and consequently the total number of leukocytes, in the exudate 5 h after carrageenin injection, as potently as dexamethasone (0.3 mg/kg) (Fig. 2).

View larger version (40K): [in this window] [in a new window]   Fig. 1.   Effects of FR167653 (FR; 3, 10, 30 mg/kg) and dexamethasone (DEX; 0.3 mg/kg) on pleural exudate volume (upper panel) and the amount of dye exuded over 20 min (lower panel) 5 h after carrageenin injection. Each value indicates the mean ± S.E.M. of 4 to 11 rats.

View larger version (30K): [in this window] [in a new window]   Fig. 2.   Effects of FR167653 (FR; 30 mg/kg) and dexamethasone (DEX; 0.3 mg/kg) on numbers of total leukocytes (top panel), neutrophils (middle panel), and mononuclear leukocytes (bottom panel) in the pleural exudate 5 h after carrageenin injection. Each value indicates the mean ± S.E.M. of five to seven rats.

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View larger version (23K): [in this window] [in a new window]   Fig. 3.   Effects of FR167653 (FR; 30 mg/kg) and dexamethasone (DEX; 0.3 mg/kg) on levels of prostanoids at 5 h after carrageenin injection. Each value indicates the mean ± S.E.M. of 8 to 19 rats.

View larger version (29K): [in this window] [in a new window]   Fig. 4.   A, Western blot analysis showing the effect of FR167653 (FR; 30 mg/kg) and dexamethasone (DEX; 0.3 mg/kg) on the levels of cyclooxygenase (COX)-1 (upper panel) and COX-2 (lower panel) in pleural exudate cells 5 h after carrageenin injection. The position of the 70-kDa molecular marker protein is indicated. Similar results were obtained from two additional sets of experiments. B, reverse transcription-competitive PCR analysis showing the effect of FR167653 (FR; 30 mg/kg) and dexamethasone (DEX; 0.3 mg/kg) on the level of COX-2 mRNA in pleural exudate cells at 5 h after carrageenin injection. The results were normalized by the level of -actin mRNA. A dotted line in the panel indicates the detection limit for COX-2 mRNA. Each value indicates the mean ± S.E.M. of three to six rats.

View larger version (31K): [in this window] [in a new window]   Fig. 5.   A, time course of the levels of tumor necrosis factor- (closed circles with solid line) and interleukin-1 (open circles with broken line) in the pleural exudate. Each value indicates the mean ± S.E.M. of four to six rats. B, effects of FR167653 (FR; 30 mg/kg) and dexamethasone (DEX; 0.3 mg/kg) on levels of tumor necrosis factor- (left panel) and interleukin-1 (right panel) 2 h after carrageenin injection. Each value indicates the mean ± S.E.M. of four to six rats.

Effects in the Late Phase of Pleurisy. We have previously demonstrated that a high level of cyclooxygenase-2 is detectable in the cells scraped off the parietal pleura 14 h after the induction of pleurisy (Hatanaka et al., 1999). These results were confirmed in the present study (Fig. 6). Dexamethasone administered 9 h after the induction inhibited the cyclooxygenase-2 expression in the scraped cells, but FR167653 did not. In addition, dexamethasone significantly suppressed 6-keto-prostaglandin F₁ levels, but FR167653 did not (Fig. 7).

View larger version (40K): [in this window] [in a new window]   Fig. 6.   Western blot analysis showing the effect of FR167653 (FR; 30 mg/kg) and dexamethasone (DEX; 0.3 mg/kg) on the levels of cyclooxygenase (COX)-1 (upper panel) and COX-2 (lower panel) in cells scraped from the parietal pleura 14 h after carrageenin injection. The position of the 70-kDa molecular marker protein is indicated. Similar results were obtained from two additional sets of experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Effects of FR167653 (FR; 30 mg/kg) and dexamethasone (DEX; 0.3 mg/kg) on levels of prostanoids at 14 h after carrageenin injection. Each value indicates the mean ± S.E.M. of 6 to 11 rats.

View larger version (83K): [in this window] [in a new window]   Fig. 8.   Hematoxylin-eosin-stained cross-sections of parietal pleura from a normal control rat (panel A), a rat after 24 h of pleurisy (panel B), and rats after 24 h of pleurisy treated with FR167653 (30 mg/kg doses injected at 9, 15, and 21 h after carrageenin injection; panel C) or dexamethasone (0.3 mg/kg doses injected at 8, 14, and 20 h after carrageenin injection; panel D). The thickness of the pleura is indicated by arrowheads. The photomicrographs show a representative section from three rats for each experiment.

Effects on Mediator-Induced Plasma Exudation. Intradermal injection of bradykinin (0.1-10 nmol/site) and histamine (0.5-500 nmol/site) caused dose-dependent amounts of plasma exudation in the rat. Dexamethasone significantly suppressed the plasma exudation induced by both mediators. However, FR167653 did not suppress it, except at the threshold dose of bradykinin (Fig. 9).

View larger version (17K): [in this window] [in a new window]   Fig. 9.   Effect of FR167653 (30 mg/kg, closed triangle) and dexamethasone (0.3 mg/kg, closed rectangles) in comparison with nontreated control (closed circles) on the plasma exudation induced by bradykinin (left panel) and histamine (right panel). Open circles indicate the amount of exuded dye induced by injection of Tyrode's solution, which is vehicle of mediators. Each value indicates the mean ± S.E.M. of 3 to 10 experiments.

	Discussion

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We performed a pharmacological characterization of FR167653 in relation to the early and late phases of rat carrageenin-induced pleurisy and mediator-induced plasma exudation in comparison with dexamethasone. Glucocorticoids up- or down-regulate the expression of a wide variety of genes (Barnes, 1998; McKay and Cidlowski, 1999). In fact, even though we chose a lower dose of dexamethasone (Kawamura et al., 2000), the drug suppressed all of the changes that we tested here, except for the leukocyte cyclooxygenase-2 expression in the early phase. In contrast, FR167653 exhibited its effects, including those on mediator-induced plasma exudation, only in the early phase, not in the late phase.

In recent years, an increasing number of compounds have been implicated in the suppression of cytokine synthesis (Lee et al., 1993). SK&F 86002 [6-(4'-pyridyl)-2,3-dihydroimidazo-(2,1-b)-thiazole, a pyridinyl imidazole], is the prototype of a novel class of cytokine synthesis inhibitors that bind CSBP kinase and inhibit its activity (Lee et al., 1993, 1994). In the present study, FR167653 significantly suppressed the levels of tumor necrosis factor- and interleukin-1 (Fig. 5B). Thus, FR167653 showed a cytokine synthesis inhibitory activity in this acute exudative inflammatory model as potent as in human alveolar macrophages and peripheral blood monocytes (Kawano et al., 1999), in a rat lipopolysaccharide-induced disseminated intravascular coagulation model (Yamamoto et al., 1996), and in a rat lipopolysaccharide-induced lung injury model (Yoshinari et al., 2001). Recently, it is reported that FR167653 reduces the expression of p38 mitogen-activated protein kinase (Otani et al., 2000) and the phosphorylation of p38 mitogen-activated protein kinase (Yoshinari et al., 2001) in the rat lung tissue. Thus, FR167653 may exert anti-inflammatory effects interfering with p38 mitogen-activated protein kinase pathway.

We showed here that dexamethasone suppressed cyclooxygenase-2 expression in mesothelial cells (Fig. 6) but did not affect that in leukocytes (Fig. 4). We have previously reported that a lower anti-inflammatory dose of dexamethasone (0.3 mg/kg) did not affect the level of cyclooxygenase-2 protein expressed in leukocytes in this model, whereas higher doses of the drug suppressed it (Kawamura et al., 2000). The present results confirm the profile of the lower dose of dexamethasone on leukocyte cyclooxygenase-2 expression in the levels of protein (Fig. 4A) and mRNA (Fig. 4B). In contrast, FR167653 suppressed cyclooxygenase-2 expression in leukocytes (Fig. 4) but did not affect that in mesothelial cells (Fig. 6). These are similar to the results seen with SB203580 [4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)-imidazole], another pyridinyl imidazole class of cytokine synthesis inhibitor that inhibits murine macrophage cyclooxygenase-2 expression, whereas the inhibitor had no significant effect on cyclooxygenase-2 expression in bovine chondrocytes (Patel et al., 1999). Furthermore, FR167653 lowered the level of cyclooxygenase-2 protein, but did not significantly affect the cyclooxygenase-2 mRNA level in leukocytes (Fig. 4), suggesting the involvement of translational events. In contrast, the drug lowered the cyclooxygenase-2 mRNA level in lipopolysaccharide-stimulated human peripheral blood monocytes and alveolar macrophages, suggesting the involvement of transcriptional or mRNA stability events or both (Kawano et al., 1999). In relation to the contrasting results obtained by us for the effects of FR167653 on the cyclooxygenase-2 mRNA level, SK&F 86002 suppressed expression of tumor necrosis factor- and interleukin-1 at a translational level in human peripheral blood monocytes stimulated with lipopolysaccharide (Lee et al., 1993; Young et al., 1993) and the THP-1 human monocytic cell line stimulated with lipopolysaccharide (Prichett et al., 1995). However, it caused a decrease in the cyclooxygenase-2 mRNA level in human peripheral blood monocytes stimulated with serum-treated zymosan (Pouliot et al., 1997). Therefore, the level at which proinflammatory gene expression is affected by this class of cytokine synthesis inhibitors is still controversial. The level would vary depending on cell types and on the circumstances that cells encounter.

Tumor necrosis factor- and interleukin-1 were detectable in the pleural exudate (Fig. 5A). In addition, intrapleural injection of tumor necrosis factor- induces cyclooxygenase-2 expression in leukocytes (Hatanaka et al., 1996). These facts suggest that tumor necrosis factor- may be a candidate for inducing cyclooxygenase-2 in this model. FR167653 suppressed the expression of cyclooxygenase-2 (Fig. 4A) as well as tumor necrosis factor- and interleukin-1 (Fig. 5B). Pyridinyl imidazoles suppress cyclooxygenase-2 expression induced by tumor necrosis factor- and interleukin-1 (Pouliot et al., 1997). Therefore, it may well be that FR167653 suppresses cyclooxygenase-2 expression through direct interference with cyclooxygenase-2 gene expression but not by blocking cytokine production.

In the early phase, FR167653 lowered the measured levels of prostanoid (Fig. 3). In contrast, the drug did not affect the prostanoid levels in the late phase (Fig. 7). This result suggests that FR167653 may not directly interfere with enzymes involved in prostanoid formation, whereas SK&F 86002 was originally reported to be a dual inhibitor of arachidonic acid metabolism, since it inhibits both cyclooxygenase and 5-lipoxygenase (Griswold et al., 1987). We have previously shown that selective cyclooxygenase-2 inhibitors suppress preferentially the level of prostaglandin E₂, but they did not significantly lower the levels of thromboxane B₂ and 6-keto-prostaglandin F₁ in the early phase of this model (Harada et al., 1996). However, FR167653 and dexamethasone almost equally lower the levels of these prostanoids (Fig. 3). Thus, there exists a difference in profiles at each prostanoid level. Both FR167653 and dexamethasone inhibited intrapleural infiltration of leukocytes (Fig. 2), which may contribute to prostanoid formation in the inflammatory site. Therefore, the inhibitory effect of FR167653 on the leukocyte infiltration may partly contribute to the lowering of the prostanoid level. In addition, FR167653, and higher doses of dexamethasone, suppress cyclooxygenase-2 protein expression (Fig. 4A; Kawamura et al., 2000). Murakami et al. (1997) proposed that intracellular translocation of enzymes involved in prostanoid formation may occur after induction of cyclooxygenase-2. These mechanisms might explain the difference in inhibitory profiles between selective cyclooxygenase-2 inhibitors and FR167653.

Both FR167653 and dexamethasone lowered the level of prostanoids and suppressed plasma exudation in the early phase (Figs. 1 and 3). In carrageenin-induced pleurisy, bradykinin (Majima et al., 1993) and prostanoids, especially prostaglandin E₂ (Harada et al., 1998), play a crucial role in plasma exudation. Prostanoid itself does not induce plasma exudation but potentiates that induced by bradykinin or other mediators (Williams and Morley, 1973). Dexamethasone suppressed mediator-induced plasma exudation, but FR167653 did not consistently affect it (Fig. 9). These results suggest that FR167653 may exhibit an inhibitory effect on plasma exudation through lowering the level of prostanoids, especially prostaglandin E₂, in the inflammatory site.

Neutrophils are activated by many chemotactic factors. Proinflammatory cytokines, such as tumor necrosis factor-, interleukin-1, and interleukin-8, also induce neutrophil activation (Cybulsky et al., 1986; Utsunomiya et al., 1996). These cytokines were detectable in this model, and FR167653 suppressed levels of tumor necrosis factor- and interleukin-1 (Fig. 5). FR167653 is also reported to suppress the level of interleukin-8 (Aiba et al., 2000). It is demonstrated that dexamethasone suppresses mediator-induced leukocyte infiltration (Katori et al., 1990). On the other hand, SK&F 86002 does not inhibit LTB₄-induced leukocyte chemotaxis, whereas it does inhibit inflammatory cell infiltration induced by carrageenin in the mice (Griswold et al., 1989). All these results suggest that this class of cytokine synthesis inhibitors may inhibit leukocyte infiltration through inhibition of the production of mediator(s), which activate leukocytes, but not through leukocyte migration itself.

FR167653 suppressed plasma exudation and leukocyte infiltration in the early phase of inflammation, as did dexamethasone (Figs. 1 and 2). However, it did not suppress the expression of cyclooxygenase-2 or hyperplasia of the pleural mesothelium in the late phase (Figs. 6 and 8). It is suggested that prostaglandin I₂ may be generated via cyclooxygenase-2 and be involved in the hyperplasia of pleural mesothelium, because selective cyclooxygenase-2 inhibitors lowered the level of 6-keto-prostaglandin F₁ and suppressed the hyperplasia, simultaneously (Hatanaka et al., 1999). Dexamethasone suppressed the cyclooxygenase-2 expression and preferentially lowered 6-keto-prostaglandin F₁ (Figs. 6 and 7). As mentioned above, FR167653 suppressed the cyclooxygenase-2 expression and lowered almost equally all prostanoids measured (Figs. 3 and 4A). These results suggest the presence of a difference between leukocytes and mesothelial cells in regulatory mechanism of prostanoid formation. The role of pleural hyperplasia in the late phase of inflammatory response is not clear, but it may participate in the healing process (Mizuno et al., 1997; Shigeta et al., 1998).

FR167653 exhibited potent anti-inflammatory effects and suppressed proinflammatory gene expression, especially in the early phase of carrageenin-induced pleurisy. The selective suppression of gene expression may be beneficial in a safer anti-inflammatory agent. Thus, FR167653 may be the source of new therapeutic candidates with a spectrum of activity distinct from current anti-inflammatory steroids.