Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

The cytokines play various biological roles in homeostasis, defense mechanisms, and immune regulation. One multifunctional cytokine, IL-6 (Akira et al., 1990), is involved in immune reaction regulation (Matsuda et al., 1989), hematopoiesis (Eaves et al., 1991), acute phase response (Dowton et al., 1991), and growth of certain types of tumor cells (Roodman, 1997). However, its excessive production plays a major role in cancer cachexia (Strassmann et al., 1992), Castleman's disease (Yoshizaki et al., 1989), rheumatoid arthritis (Takagi et al., 1998), hypercalcemia (Schweitzer et al., 1995), and multiple myeloma (Zhang et al., 1990; Roodman, 1997). Therefore, modulation of this cytokine function may be potentially effective against cancer and chronic or refractory diseases.

Recent studies have revealed various possibilities for control of IL-6 activity. IL-6, a four (A, B, C, and D)-helix bundle cytokine, is believed to interact sequentially via distinct binding sites with two transmembrane receptors: the low-affinity IL-6R and the signal transducer 130-kDa glycoprotein (gp130). Savino et al. (1994) generated bifacial mutations that were combined with amino acid substitution in predicted D helix that increase binding for IL-6R. The mutant has no biological activity, but it improved first receptor occupancy and fully inhibits the wild-type cytokine at low dosage on a variety of human cell lines. Recently, IL-6 has been shown to possess three topologically distinct receptor binding sites: site 1 for binding to the subunit-specific chain IL-6R, and sites 2 and 3 for interaction with two subunits of the signaling chain gp130. Sporeno et al. (1996) generated a set of IL-6 variants that behave as potent cytokine receptor antagonists carrying substitutions that abolish interaction with gp130 at either site 2 alone (site 2 antagonist) or at both sites 2 and 3 (site 2 + 3 antagonist). Site 2 antagonists were generally quite effective, only the site 2 + 3 antagonist showed antagonism on the full spectrum of cells tested.

Ward et al. (1996) also suggested that a stable IL-6 dimer mutant may be an efficient IL-6 antagonist. Furthermore, Sato et al. (1996) successfully humanized a mouse monoclonal antibody by glycosylation in its heavy chain variable region, which specifically binds to IL-6 and strongly inhibits IL-6 functions.

As mentioned, by development of high molecular biocompounds such as various IL-6 variants or humanized antibody, it was indicated that there is a possibility to control activity and production of IL-6 selectively and in low concentration. Consequently, for clinical application, development of low molecular antagonist is anticipated because of superiority in oral absorbency, antigenicity, and so forth.

We have discovered many new bioavailable compounds (Hayashi et al., 1995; Sunazuka et al., 1997; Fukami et al., 2002) with unique action mechanisms from natural products. In a previous study, we found madindolines (Hayashi et al., 1996; Sunazuka et al., 2000) having gp130 antagonism in research for IL-6 inhibitors from microbial metabolites (Hayashi et al., 2002). As a result of screening from natural products in searching for a stronger IL-6 inhibitor, we found 20S,21-epoxy resibufogenin-3-formate (ERBF; Fig. 1) (Kamano et al., 2002) from bufodienolide. In this study, along with examining peculiarities of ERBF by cell proliferation, expression, and morphological change inducted by various cytokines as indicators, we investigated its function as a receptor and specific signal transduction cascade and analyzed its action mechanism.

View larger version (10K): [in this window] [in a new window]   Fig. 1.   Structure of ERBF.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

Materials. Sigma-Aldrich (St. Louis, MO) was the vendor for rmIL-2, rmIL-3, rhIL-4, rhIL-8, rm tumor necrosis factor (TNF), rhsIL-6R, and rhIL-11; rhIL-6, rh leukemia inhibitory factor (LIF), recombinant human nerve growth factor (NGF), and 1,25(OH)₂D₃ (carcitrial) were purchased from Upstate Biotechnology (Lake Placid, NY), Toyo (Tokyo, Japan), and Wako Pure Chemicals (Tokyo, Japan), respectively. Monoclonal anti-IL-8 antibody and FITC-conjugated anti-CD23 antibody were purchased from Seragen, Inc. (Hopkinton, MA). MH-60 cells and CTLL-2 cells were kindly supplied by Prof. Hirano (Osaka University, Osaka, Japan) and Prof. Ishizuka (Microbial Institute, Shizuoka, Japan), respectively. Baf3 cells, L929 cells, U937 cells, and PC-12 cells were kindly gifted from the Health Science Research Resources Bank (Osaka, Japan).

Cytokine-Dependent Cell Growth. CTLL-2 cells, Baf3 cells, and IL-6-dependent MH-60 cells were maintained in suspension in RPMI 1640 medium supplemented with 10% fetal calf serum (FCS) containing 1 ng/ml rmIL-2, 1 ng/ml rmIL-3, or 0.5 ng/ml rhIL-6, respectively. IL-6-independent MH-60 cells were obtained by repeated cultivation of IL-6-dependent MH-60 cells in decreased concentration of IL-6 and were maintained in an RPMI 1640 medium supplemented with 10% FCS in absence of IL-6. Cells (0.2-0.5 × 10⁴ cells) suspended in 200 µl of the medium containing corresponding cytokine were plated in a 96-well culture plate (Corning, Palo Alto, CA) and incubated at 37°C in a 5% CO₂, 95% air atmosphere; 5 µl of various concentrations of ERBF was added to each well. After 72-h incubation, cell growth was measured by the tetrazolium salt method (Carmichael et al., 1987).

Flow Cytometry Analysis. U937 cells were collected, washed, and resuspended in 50 µl of cold phosphate-buffered saline () buffer. Subsequently, cells were incubated on ice with 1 µg of FITC-conjugated anti-human CD23 monoclonal antibody. After centrifugation, cells were washed and resuspended in 500 µl of cold phosphate-buffered saline () buffer and then they were analyzed by flow cytometry using EPICS ELITE (Beckman Coulter, Inc., Fullerton, CA) equipped with a 488-nm argon laser.

Neuronal Differentiation. Rat phenochromocytoma PC-12 cells were maintained in a monolayer in RPMI 1640 medium supplemented with 10% FCS. Cells (1 × 10⁴ cells) suspended in 200 µl of the medium were plated in a 96-well flat culture plate (Corning) and incubated at 37°C in a 5% CO₂, 95% air atmosphere; 5 µl of NGF (final 50 ng/ml) or IL-6 (final 3 ng/ml) was added to each well in presence or absence of 20 or 5 µM ERBF, respectively. After 72-h incubation, PC-12 cell morphological change was observed by optical microscopy.

Chemotaxis. Human polymorphonuclear leukocytes (PMNLs) were obtained from healthy donors by one-step discontinuous Percoll gradient centrifugation as described previously (Del Buono et al., 1989). Purified PMNLs (>95% purity and viability) were washed twice after lysis of contaminating erythrocytes with ice-cold ammonium chloride and resuspended in Hanks' balanced salt solution buffer. The PMNLs (1 × 10⁶ cells) were stained by 3 µM carboxyfluorescein diacetate duccinimyl ester for 20 min. Chemotactic activity was estimated by chemotaxis chamber (Ieda Trading Co., Tokyo, Japan). Carboxyfluorescein diacetate duccinimyl ester-labeled PMNLs (1 × 10⁵ suspended in 200 µl) were added to the upper chamber and 20 ng/ml rhIL-8 was added to the lower chamber, respectively. After 4-h incubation at 37°C, the reaction mixture in the lower chamber was centrifuged, and residue fluorescence intensity was measured by fluorometry.

Receptor Binding Assay. This assay used sIL-6R antibody-precoated plates (sIL-6R assay kit; Bio-Rad, Hercules, CA). Fifty nanograms per 100 µl of sIL-6R was added to the plate and then incubated for 20 min at 37°C; the plate was then washed twice with assay buffer. Precoated plates were blocked by 0.25% bovine serum albumin for 20 min at 37°C and washed three times; 5 µg/ml of sample was added to the well and incubated for 20 min. Next, the plate was washed three times, and 1000 ng/100 µl of IL-6 was added to the well and incubated for 10 min at 37°C. After centrifugation for 5 min at 800 rpm at 4°C, the supernatant was assayed for unbound (free) IL-6.

Coculture of Osteoblastic Cell and Bone Marrow Cells. Coculture of mouse calvaria cells (osteoblastic cells) and bone marrow cells was performed by the method of Tamura et al. (1993) using ddY mice. Briefly, primary osteoblast-like cells (1 × 10⁴/well) obtained from mouse calvaria and nucleated bone marrow cells (2 × 10⁵/well) were cocultured in the 48-well plate with 0.4 ml of -minimal essential medium containing 10% FCS and 20 ng/ml rhIL-6 (containing 50 ng/ml rhIL-6R) or 10 ng/ml rhIL-11 in presence or absence of ERBF. Cultures were performed in duplicate on 8 days. To determine the number of osteoclasts formed, cells were fixed and stained for tartrate-resistant acid phosphatase (TRAP), and the number of TRAP-positive osteoclasts was counted.

Action Mode Analysis. Inhibitory mode of ERBF for IL-6 activity was statistically analyzed by Schild plot analysis. EC₅₀ in the dose-response curve of A alone is shown as [Ao], EC₅₀ in the dose-response curve of A with coexisting B is depicted as [Ax], and concentration of existing B is [Bx]. The dissociation constant of B and the receptor combined is KB. The formula for these relations can be expressed as log ([Ax]/[Ao]  1) = log [Bx]  log KB. Using this formula, we plotted log ([Ax]/[Ao]  1) on the y-axis against log [Bx] on the x-axis.

Statistical Analysis. Statistical significance of differences between the control and the experimental group was determined by Student's t test.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

As shown in Fig. 2, ERBF suppressed IL-6 activity and caused a parallel rightward shift of dose-response curves to IL-6 at concentrations of 0.03 to 10 ng/ml. Therefore, it seems that there is significant difference between 0.03 and 10 ng/ml of IL-6 and those in presence of 7.5, 15, and 30 µM ERBF, respectively. Analysis of data yielded a pA₂ value of 5.12 and a slope of 0.99. The slope did not differ significantly from unity.

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Effect of ERBF on IL-6-dependent cell growth and Schild plot analysis. A, MH60 cells were incubated with various doses of rhIL-6 in presence or absence of ERBF: 0 µM (open squares), 7.5 µM (open diamonds), 15 µM (open circles), and 30 µM (open triangles). B, y-axis values and x-axis values show log ([AX]/[Ao]  1) and log [ERBF], respectively. The data yield a slope of 0.99.

Suitable concentrations of various cytokines were examined before examination of ERBF. Maximum cell growth of CTLL-2 cells, Baf3 cells, and MH-60 cells was induced by 20 ng/ml IL-2, 5 ng/ml IL-3, and 2 ng/ml IL-6, respectively.

We investigated whether ERBF specifically inhibits growth of cells induced by cytokines. Treatment with 70 µM ERBF did not change submaximal growth of IL-2-dependent CTLL-2 cells (Fig. 3A). Similarly, ERBF (even at 70 µM) did not suppress maximal cell growth of IL-3-dependent Baf3 cells at a concentration of 5 ng/ml (Fig. 3B). However, ERBF inhibits activity of 2 ng/ml IL-6, which induces submaximal growth of IL-6-dependent MH-60 cells, in a dose-dependent manner (Fig. 3C); in contrast, the same dose of ERBF did not show any growth inhibition of IL-6-independent MH-60 cells. TNF (2 U/ml)-induced growth inhibition on mouse fibrosarcoma L929 cells was not overcome by ERBF (Fig. 3D).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Effect of ERBF on activities of IL-2, IL-3, IL-6, and TNF. IL-2-dependent CTLL-2 cells (open squares), IL-3-dependent Baf3 cells (closed squares), and IL-6-dependent (closed circles) or -independent (open circles) MH60 cells were incubated with various concentrations of ERBF in presence of IL-2 (20 ng/ml) (A), IL-3 (5 ng/ml) (B), or IL-6 (2 ng/ml) (C), respectively. TNF-sensitive L929 cells were incubated with ERBF in presence (closed triangles) or absence (open triangles) of TNF (2 U/ml) (D).

To study whether ERBF inhibits activity of other cytokines, we examined surface antigen FcR II (CD23) expression caused by 10 ng/ml rhIL-4 in U937. With treatment of 10 and 70 µM, however, ERBF showed no suppression of antigen expression (Fig. 4, A-D).

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Effect of ERBF on expression of FcR II (CD23) on IL-4-stimulated U937 cells. U937 cells were incubated with 0 (A) or 10 ng/ml (B) rhIL-4 in absence or presence of 10 µM ERBF (C) or 70 µM ERBF (D). Cells were stained by FITC-conjugated anti-CD23 monoclonal antibody and then analyzed by flow cytometry.

The ERBF effect on NGF activity was examined in NGF-induced neuronal differentiation. As reported by Lazarini et al. (1994), PC-12 cells extended neurite outgrowth when treated with either 50 ng/ml recombinant human NGF or 30 ng/ml rhIL-6 and formed neuronal networks as shown in Fig. 5, A and C, respectively. Even at 20 µM, ERBF did not affect NGF-induced neuronal differentiation (Fig. 5B), whereas ERBF at 5 µM completely inhibited IL-6-induced differentiation (Fig. 5D), suggesting that ERBF has no effect on the action of NGF.

View larger version (170K): [in this window] [in a new window]   Fig. 5.   Effect of ERBF on neuronal differentiation. PC-12 cells were incubated for 72 h at 37°C with 50 ng/ml NGF in absence (A) or presence (B) of 20 µM ERBF. The PC-12 cells were also incubated with 3 ng/ml rhIL-6 in absence (C) or presence (D) of 5 µM ERBF.

The effect of ERBF on IL-8 activity was further examined in chemotaxis of human neutrophil (Table 1). Fluorescence intensity in the lower chamber increased when 20 ng/ml rhIL-8 was added to the medium of the lower chamber; also, the increase was suppressed by addition of monoclonal anti-IL-8 antibody to the lower chamber but not to the upper chamber. This implies chemotaxis of FITC-labeled PMNLs from the upper chamber to the lower chamber. ERBF (20 µM) did not change the IL-8-induced increase in fluorescence in the lower chamber when added to both chambers. This result suggests that ERBF showed neither chemotactic activity nor suppression of IL-8 activity.

Next, we investigated ERBF efficacy on formation of osteoclasts used as an indicator for activity of IL-6-type cytokines (Fig. 6). In a coculture of mouse carvalia osteoblast cells and bone marrow cells, IL-6, IL-11, LIF, and 1,25(OH)₂D₃ (as positive control) induced in marked TRAP-positive multinuclear cells (osteoclasts). Treatment with ERBF did not affect IL-11-, LIF-, and 1,25(OH)₂D₃-induced osteoclast formation. In contrast, IL-6-induced osteoclast formation was dose dependently inhibited by ERBF and completely suppressed at 10 µM. Meanwhile, madindoline A dose dependently suppressed both IL-6- and IL-11-induced osteoclast formation, but not LIF- and 1,25(OH)₂D₃-induced formation.

View larger version (22K): [in this window] [in a new window]   Fig. 6.   Effect of ERBF and MDL-A on osteoclast formation induced by several cytokines that share gp130 as a common signal transducer or 1,25(OH)2D3. Mouse calvaria cells (osteoblastic cells, 1 × 104 cells/well) and bone marrow cells (2 × 105 cells/well) were cocultured with -minimal essential medium containing 20 ng/ml IL-6 (containing 50 ng/ml soluble IL-6R) (closed squares), 20 ng/ml IL-11 (closed diamonds), 50 ng/ml LIF (closed circles), or 5 µM 1,25(OH)2D3 (closed triangles) in presence or absence of ERBF (A) or MDL-A (B). After 8 days of incubation, the number of TRAP-positive multinuclear cells (osteoclasts) was counted. Bars represent mean ± S.E. of three or four experiments.

The effect of ERBF on IL-6 receptor was evaluated by measurement of unbound IL-6 to the receptor after incubation of IL-6R with IL-6. As shown in Table 2, amounts of unbound IL-6 to the receptor increased approximately 20-fold by pretreatment with anti-IL-6R antibody used as a positive control. Similarly, pretreatment with ERBF caused dose-dependent increases in unbound IL-6, whereas madindoline A (MDL-A) did not increase in unbound IL-6.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Recent studies reveal the distinct relationship between IL-6 receptor subunits and many refractory diseases, and the importance of IL-6 inhibitor for prevention and therapy of the diseases. Novick et al. (1989) and Honda et al. (1992) report that naturally produced sIL-6R occurs in urine and sera of healthy subjects, and that its serum levels are increased in patients with multiple myeloma (Gaillard et al., 1993), cancer cachexia (Strassmann and Kambayashi, 1995), and human immunodeficiency virus infection (Honda et al., 1992). Furthermore, Lu et al. (1995) and Hitzler et al. (1991) report that excess IL-6 is produced systemically and locally in patients with multiple myeloma. Symons et al. (1989) and Uson et al. (1997) also report that both IL-6 and sIL-6R levels were elevated significantly in synovial fluids from patients with rheumatoid arthritis. Lin et al. (1997) demonstrate that sensitivity of the osteoclastogenic process to this cytokine is altered after ovariectomy and that ovariectomy in mice caused an increase in gp80, gp130, and IL-6 mRNA expression in bone marrow cell cultures. These observations raise the possibility that the number of IL-6Rs on the cell membrane or concentration of sIL-6R in body fluids may be important factors controlling IL-6 response in target tissues in physiological and/or pathological conditions. The present study clearly demonstrates that ERBF did not affect any activity of various cytokines except IL-6 and is a first specific small molecule to show IL-6 receptor antagonistic activity. ERBF would thus be useful as an inhibitor of IL-6R for treatment of metabolic bone diseases.

A shared signal-transducing receptor subunit can be recruited by different cytokines and, depending upon the ligand, be activated via homodimerization or heterodimerization with other cytokine receptors. Three shared receptor subunits have been cloned so far: the -chain commonly used by IL-2, IL-4, IL-7, IL-9, and IL-15 (IL-2-type cytokines); the -chain common to IL-3, IL-5, and granulocyte-macrophage colony stimulating factor (IL-3-type cytokines); and gp130, which is shared by IL-6, IL-11, LIF, ciliary neurotrophic factor, Oncostatin M (OSM), and cardiotrophin-1 (IL-6-type cytokines). In our experiments, ERBF did not suppress cell proliferation, differentiation, expression, and chemotaxis of any tested cytokines and growth factors except IL-6. Our results clearly indicate that ERBF does not affect either the -chain common to IL-2-type cytokines or the -chain common to IL-3-type cytokines; also, ERBF is a small molecule showing highly selective inhibitory and noncytotoxic activities.

The IL-6 exerts its activity via a cell surface receptor consisting of two components: a ligand-binding 80-kDa IL-6R, and a signal-transducing 130-kDa gp130. IL-6 induces gp130 dimerization after binding to the IL-6R, which leads to activation of the Janus kinase/STAT signal transduction pathway (Heinrich et al., 1998). Subsequently, phosphorylated STATs dimerize and translocate into the nucleus to activate expression of target genes (Lutticken et al., 1994). Therefore, it is considered that the active site of ERBF is either 1) a binding between IL-6 and IL-6R, 2) a complex formation of IL-6/IL-6R and gp130, 3) a homodimerization of gp130, 4) a signal transduction of Janus tyrosine kinase/STAT, or 5) the expression of target genes.

IL-6-type cytokines either signal through gp130 alone or in combination with the LIF receptor or the recently cloned OSM receptor. IL-6 and IL-11 induce gp130 homodimerization (Yin et al., 1993), whereas ciliary neurotrophic factor (Davis et al., 1993), LIF (Gearing et al., 1991), OSM (Gearing et al., 1992), and cardiotrophin-1 (Pennica et al., 1995) signal via heterodimerization of gp130 and LIF receptor (Davis et al., 1993). ERBF did not inhibit osteoclastogenesis induced by both LIF (heterodimer type) and IL-11 (homodimer type). In contrast, madindoline A, a gp130 homodimerization inhibitor, dose dependently suppressed osteoclast formation induced by IL-6 and IL-11, which are shared homodimer types of gp130. These results suggest that ERBF acts on the extracellular domain of IL-6 signal, not homo- or heterodimerization of gp130 and intracellular signal transduction cascade.

In receptor binding assay, ERBF increased the unbound IL-6 in a dose-dependent manner, suggesting that ERBF suppresses binding of IL-6 to IL-6R (Table 2). Schild plot analysis can be used to identify competitive interactions between drugs. If an antagonist produces dose-related, parallel, rightward displacements of an antagonist dose-effect function, and if the slope of a Schild regression does not differ from unity, the relationship between drugs is presumed to be a competitive interaction and the resultant pA₂ value provides an estimate of the KB of the antagonist. EC₅₀ in the dose-response curve of A alone is shown as [Ao]; EC₅₀ in the dose-response curve of A with coexisting B is depicted as [Ax], and concentration of existing B is [Bx]. The dissociation constant of B and the receptor combined is KB. The formula for these relations can be expressed as log ([Ax]/[Ao]  1) = log [Bx]  log KB. This formula was used to plot log ([Ax]/[Ao]  1) on the y-axis against log [Bx] on the x-axis. Both agonist A and antagonist B are expressed with a "1" numerator. Linearity of slope 1 is expressed only in the competitive case. Moreover, log ([Ax]/[Ao]  1) = log [Bx]  log KB indicates that when the y-axis value is 0, the x-axis value is pA₂ (log KB). In this analysis, the linear curve slope is 0.99; according to results of the Fig. 2A Schild plot; it is almost straight with a near-unity slope value. Therefore, it is confirmed that ERBF functions by competitive interaction. In addition, its pA₂ value is 5.12.

It has been suggested that excess production of IL-6 is closely related to progression of hormone-dependent hypercalcemia (Schweitzer et al., 1995) and development of multiple myeloma (Roodman, 1997) and rheumatoid arthritis (Takagi et al., 1998). Although high molecular biocompounds, such as various IL-6 variant or humanization of mouse antibody have been discovered, when clinical applications are considered, development of a low molecular antagonist is anticipated because of superiority in oral absorbency, antigenicity, cost, and so forth. IL-6 inhibitors of low molecular weight such as ERBF may be useful in studying the role of IL-6 in disease. In summary, ERBF is a specific and potent inhibitor of IL-6 activity. In view of in vitro activity, it would be of interest to examine the efficacy of ERBF in vivo in therapy against refractory diseases such as cancer cachexia, postmenopausal osteoporosis (Papadopoulos et al., 1997), and chronic inflammatory disease.