A small synthetic molecule capable of preferentially inhibiting the production of the CC chemokine monocyte chemotactic protein-1.

ARTICLE

INTRODUCTION

Chemokines are a superfamily of cytokines which play a crucial role in inflammation, infection and immunity [1-3]. Monocyte chemotactic protein-1 (MCP-1) is a CC chemokine active on monocytes, activated T lymphocytes and NK cells [1, 4]. MCP-1 is involved in the regulation of monocyte recruitment in a variety of pathological conditions, including atherosclerosis, neoplasia, immuno-inflammatory diseases and HIV infection [1, 4]. Blocking chemokine production or action is a major target for pharmacological intervention in a variety of human diseases. While N-terminal truncations or alterations have long been known to generate peptides with antagonistic activity (e.g. MCP-1, MCP-3, RANTES) [5, 6], only recently have chemical receptor antagonists been described [7-10]. Selective inhibitors of chemokine synthesis have not been described. Bindarit, (2-methyl-2-[[1-(phenylmethyl)-1H-indazol-3yl]methoxy] propanoic acid) is a novel molecule that is devoid of immunosuppressive activity (e.g. antibody production, T cell proliferation), does not affect prostaglandin production, but inhibits adjuvant arthritis in rats [11]. Here, we report its capacity to inhibit MCP-1 production and demonstrate the in vivo relevance of these observations.

MATERIALS AND METHODS

Animals

NZB/W female mice were from Jackson Laboratory (Bar Harbor, Me, USA) and Crl:CD1(ICR)BR male mice (referred to as CD1) were from Charles River (Calco, Italy). Bindarit (from Angelini, A.C.R.A.F., Aprilia, Italy) was administered by gavage in 0.5% methylcellulose (Fluka, Buchs, Switzerland) or in a medicated diet (0.5%).

Reagents: medium RPMI 1640, (Biochrom KG, Berlin, Germany); penicillin and streptomycin for clinical use (Pharmacia, Nerviano, Italy); fetal calf serum (FCS) (Hyclone Lab., Logan, UT): all the reagents contained less than 0.125 EU/ml of endotoxin (Limulus Amebocyte Lysate assay, Microb. Associates, Walkersville, MD). LPS (from Escherichia coli 055:B5) was from Difco (Detroit, MI) and actinomycin D (AcD) from Sigma (St Louis, Mo). C. albicans was prepared as previously described [12].

Peripheral blood mononuclear cells (PBMC)

Buffy coats from blood donations (courtesy of Centro Trasfusionale, Ospedale Sacco, Milano, Italy) were used as a source of PBMC which were isolated by Ficoll-Hypaque (Biochrom KG, Berlin, Germany) gradient centrifugation. 2 x 10⁵ cells/0.2 ml in RPMI + 1% FCS were cultured in the presence of stimuli (LPS 100 ng/ml or C. albicans 0.1 mg/ml) for 24 hours in polypropylene, round-bottomed 96 wells plate (Costar Corporation, Cambridge, MA, USA).

Mono Mac 6 cells (MM6)

The MM6 line was a kind gift from Dr. Gunter Wolf (University of Hamburg, Hamburg, Germany). Cells, originally described with characteristics of mature monocytes [13], were grown in fortified RPMI1640 medium with 10% FCS as described [13]. To induce cytokine production, MM6 cells (5 x 10⁵/ml) were cultured for 24 hours in the presence or absence of LPS (100 ng/ml) in 1 ml of RPMI 1640 with 1% FCS.

Measurement of cytokines

Human MCP-1, TNF-alpha, IL-1 and IL-8 were detected using a sandwich ELISA and IL-6 was measured by a bioassay, as described [12, 14]. Human MIP-1alpha and RANTES and murine MCP-1 were measured with commercial ELISA kits, from Amersham Life Science (Buckinghamshire, England) and Benfer-Scheller (Milan, Italy), respectively.

Northern blot analysis

Northern blot analysis was performed according to standard procedures by the guanidine isothiocyanate method. Total RNA was analysed with MCP-1 and IL-8 probes prepared and used as already described [14]; RNA transfer to membranes was checked by UV irradiation, as shown in each figure. Densitometric analysis of autoradiografic signals were performed with a scanning densitometric apparatus (Hoefer, San Francisco, CA).

In vivo models

The air pouch model of local inflammation was prepared as previously described [15]. In brief, mice were anaesthetised and subcutaneous dorsal pouches were created by injection of 5 ml of sterile air. After 3 days, the pouches were reinjected with 3 ml of air. On day 6, 1 ml of 1% iota carrageenan (Sigma, St. Louis, Mo) in sterile saline or 20 ng of human IL-1ß (Dompé, L'Aquila, Italy) in sterile 0.5% carboxymethylcellulose (CMC) were injected into the pouches. The corresponding controls received sterile saline or 0.5% CMC. At selected times (4 hours for IL-1ß and 24 hours for carrageenan) the animals were sacrificed and pouches washed with 1 ml of saline. Leukocytes were stained with Diff-Quick for differential counting. Exudates were centrifuged at 5,000 rpm for 10 min at 4° C and the supernatant was stored at ­ 20° C until used. Bindarit was given daily, by gavage at dose of 50 mg/kg to two month old NZB/W mice; control mice received vehicle (0.5% methylcellulose). Treatment lasted throughout the animal's survival. Basal urinary protein excretion levels were determined before starting the treatment and values ranged from 0.3 to 2.9 mg/24 hours. During the follow up, mice showing protein levels exceeding 3 mg/24 hours were considered proteinuric. Proteinuria was measured monthly by the modified Coomassie blue G dye-binding assay for protein as described [16].

RESULTS AND DISCUSSION

To investigate the capacity of bindarit to inhibit cytokine production, human peripheral blood monocytes, major producers of MCP-1 [1, 2, 17-19], were exposed to different concentrations of the drug. As summarised in Figure 1 A and B, bindarit in vitro caused a dose-dependent inhibition of the capacity of human monocytes to produce MCP-1 in response to bacterial LPS or C. albicans. The IC₅₀ was 172 and 403 µM for LPS (15 experiments) and C. albicans (9 experiments) respectively. The production of the proinflammatory cytokines IL-1 and IL-6 was not affected by bindarit, while that of TNF-alpha by LPS-stimulated PBMC was dose-dependently inhibited (Figure 1A).

The action of bindarit on chemokine production was further investigated by studying its effects on other CC and CXC chemokines. As shown in Figure 1A, B and C, bindarit did not affect the LPS-induced production of the CXC chemokine IL-8 and of the CC chemokines MIP-1alpha and RANTES. The selective inhibitory activity of bindarit was also evident when the MM6 cell line was used (Figure 1D). Bindarit inhibited the production of MCP-1 by LPS-stimulated MM6 without affecting the release of IL-8 or IL-6. The IC₅₀ of Bindarit for MM6 cells was 425 µM, higher than monocytes. As shown in Figure 2, representative of 5 subjects, inhibition of MCP-1 production in monocytic cells was associated with reduced levels of mRNA transcripts, with an IC₅₀ of 75 µM. In agreement with protein production, bindarit did not affect the levels of IL-8 mRNA (Figure 2).

In an effort to assess the in vivo relevance of these in vitro observations, the air pouch system was used as described [15]. As shown in Figure 3A, injection of carrageenan caused recruitment of leukocytes into the air pouch fluid. Treatment with 100 mg/kg p.o., (oral LD₅₀ > 2 g/kg) caused a 60% reduction (p < 0.05) in the number of recruited leukocytes, with no diminution in the number of PMN, but with a significant reduction (50%, p < 0.05) in the number of monocytes. Concomitantly, the local production of MCP-1 was significantly reduced in bindarit-treated mice, with 35.5 ± 17.9 ng/ml of pouch fluid compared to 117.3 ± 57.3 for controls (p < 0.01). Similar results were observed when mice were treated for 17 days with the compound in the diet (0.5%) and carrageenan or IL-1 (20 ng/mouse) used as inflammatory stimuli.

It was important to assess whether the anti-MCP-1 activity of bindarit had any therapeutic potential. In a previous study, bindarit was shown to inhibit adjuvant-induced arthritis in rats [11]. Evidence for a major pathogenetic role of MCP-1 has been obtained in autoimmune kidney disorders in mice and humans [20, 21]. As shown in Figure 3B, daily oral treatment with bindarit significantly prolonged survival (p < 0.01) and delayed the onset of proteinuria (p < 0.05) in NZB/W mice, which provide a model for lupus nephritis. Similar results were obtained in parallel studies in which the compound was also administered in combination with cyclophosphamide or methylprednisolone [16, 22]. Circulating drug levels under these conditions were in the same range of concentrations as those active on MCP-1 production (see above).

Glucocorticoid hormones and anti-inflammatory cytokines (IL-4, IL-13 and IL-10) inhibit chemokine production, affecting both CC and CXC molecules [23]. Interestingly, the action of IL-4, IL-13 and IL-10 is dramatically influenced by the cellular context, since these molecules inhibit monocytes and have no effect, or stimulate endothelial cells [23]. IFN-gamma is the only agent which differentially affects members of the CC and CXC families, as it inhibits production of IL-8 but induces MCP-1 and related molecules [23, 24]. These agents have been shown to act at the level of chemokine gene transcription [19, 24]. Because the regulation of MCP-1 gene expression differs considerably from that of other chemokines, involving 5' and 3' regulatory sequences [25], heterogeneity among chemokines in the regulation of gene transcription likely underlies differential regulation by immunomodulatory agents, including the unique selective action of bindarit.

The results presented here show that, as regards chemokines, bindarit is a selective inhibitor of MCP-1 production in vitro and in vivo and suggest that its beneficial effects in models of joint and kidney inflammation are related to its anti-MCP-1 action. Therefore, our data suggest that it is possible to selectively and differentially regulate chemokines by targeting their production with small synthetic molecules.

CONCLUSION

Acknowledgements

This work has been carried out under a research contract with Consorzio Autoimmunità Tardiva C.AU.T, Pomezia, Italy, within the "Programma Nazionale Farmaci-seconda fase" of the Ministero dell'Università e della Ricerca Scientifica e Tecnologica.

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