Agonists to the A3 adenosine receptor induce G-CSF production via NF-kappaB activation: a new class of myeloprotective agents.

OBJECTIVE: The aim of this study was to evaluate the effect of CF101, a synthetic agonist to the A3 adenosine receptor (A3AR), on the production of granulocyte colony-stimulating factor (G-CSF). The ability of CF101 to act as a myeloprotective agent in chemotherapy-treated mice was tested. METHODS: CF101 was administered orally to naïve mice and its effect was studied on blood cell counts (coulter counter), serum G-CSF level (ELISA), bone marrow colony-forming cells (soft agar culture), and splenocytes' ability to produce ex vivo G-CSF. Protein extract was prepared from splenocytes and Western blot analysis was carried out to evaluate expression level of key proteins. In an additional set of experiments, CF101 was administered to mice 48 hours after cyclophosphamide treatment and blood cell counts as well as serum G-CSF levels were monitored. RESULTS: Oral administration of CF101 to naïve mice led to the elevation of serum G-CSF levels, an increase in absolute neutrophil counts (ANC), and bone marrow colony-forming cells. Splenocytes derived from these mice produced higher G-CSF level than controls. The molecular mechanisms underlying the events prior to G-CSF production included the upregulation of NF-kappaB and the upstream kinases phosphoinositide 3-kinase (PI3K), protein kinase B/Akt (PKB/Akt), and IKK. Accelerated recovery of white blood cells and neutrophil counts were observed in cyclophosphamide-treated mice following CF101 administration. CONCLUSION: CF101 induced upregulation of the PI3K/NF-kappaB pathway leading to G-CSF production, resulting in myeloprotective effect in cyclophosphamide-treated mice.

Methotrexate enhances the anti-inflammatory effect of CF101 via up-regulation of the A3 adenosine receptor expression.

Methotrexate (MTX) exerts an anti-inflammatory effect via its metabolite adenosine, which activates adenosine receptors. The A3 adenosine receptor (A3AR) was found to be highly expressed in inflammatory tissues and peripheral blood mononuclear cells (PBMCs) of rats with adjuvant-induced arthritis (AIA). CF101 (IB-MECA), an A3AR agonist, was previously found to inhibit the clinical and pathological manifestations of AIA. The aim of the present study was to examine the effect of MTX on A3AR expression level and the efficacy of combined treatment with CF101 and MTX in AIA rats. AIA rats were treated with MTX, CF101, or both agents combined. A3AR mRNA, protein expression and exhibition were tested in paw and PBMC extracts from AIA rats utilizing immunohistochemistry staining, RT-PCR and Western blot analysis. A3AR level was tested in PBMC extracts from patients chronically treated with MTX and healthy individuals. The effect of CF101, MTX and combined treatment on A3AR expression level was also tested in PHA-stimulated PBMCs from healthy individuals and from MTX-treated patients with rheumatoid arthritis (RA). Combined treatment with CF101 and MTX resulted in an additive anti-inflammatory effect in AIA rats. MTX induced A2AAR and A3AR over-expression in paw cells from treated animals. Moreover, increased A3AR expression level was detected in PBMCs from MTX-treated RA patients compared with cells from healthy individuals. MTX also increased the protein expression level of PHA-stimulated PBMCs from healthy individuals. The increase in A3AR level was counteracted in vitro by adenosine deaminase and mimicked in vivo by dipyridamole, demonstrating that receptor over-expression was mediated by adenosine. In conclusion, the data presented here indicate that MTX induces increased A3AR expression and exhibition, thereby potentiating the inhibitory effect of CF101 and supporting combined use of these drugs to treat RA.