Противовоспалительный эффект при применении препарата CF 101.

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

Abstract

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.

PMID:
17101059
[PubMed — indexed for MEDLINE]
PMCID:
PMC1794513

Free PMC Article

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Figure 1

Figure 1
Effect of CF101 and prophylactic or therapeutic MTX treatment on AIA rats. Rats were injected subcutaneously at the tail base with 100 μl of a suspension composed of incomplete Freund’s adjuvant and 10 mg/ml heat killed Mycobacterium tuberculosis. (a) Clinical score. Combined treatment with MTX (prophylactic treatment) + CF101 yield significantly lower values than treatment with each of the agents alone; also, all treatments yielded lower scores than control group. (b) The clinical score with combined therapeutic MTX treatment + CF101 was significantly lower than that in the other groups. (c, d) Morphopathological score. In AIA animals complete distruction of the cartilage and the bone, as well as severe inflammation in the hind paws, was noted. Treatment with a combination of MTX and CF101 preserved the normal features of the paw. AIA, adjuvant-induced arthritis; MTX, methotrexate.
Figure 2

Figure 2
Immunohistochemistry analysis of A3AR expression in paws from AIA rats. We conducted immunohistochemistry staining of paraffin-embedded sections of paw from MTX-treated, CF101-treated and MTX+CF101-treated AIA rats. MTX treatment induced receptor expression, and treatment with CF101 alone or in combination with MTX resulted in receptor downregulation. A3AR, A3 adenosine receptor; AIA, adjuvant-induced arthritis; MTX, methotrexate.
Figure 3

Figure 3
Ex vivo analysis of A3AR expression level in paw and PBMCs from AIA rats. (a, b) MTX treatment induced expression of A3AR mRNA and protein, and CF101 treatment resulted in receptor downregulation. (c) This was reflected in the PBMCs. (d) Expression of A2AAR was also induced in paw extracts derived AIA rats treated with MTX. A3AR, A3 adenosine receptor; AIA, adjuvant-induced arthritis; MTX, methotrexate; PBMC, peripheral blood mononuclear cell.
Figure 4

Figure 4
Effect of dipyridamole on the expression of A3AR in PBMCs from naïve rats. Dipyridamole (5 mg/kg intraperitoneally) was administered once to naïve rats and blood samples were drawn 2, 6, 24 and 48 hours after dipyridamole administration. Dipyridamole induced A3AR upregulation in PBMCs derived from treated rats. A3AR, A3 adenosine receptor; PBMC, peripheral blood mononuclear cell.
Figure 5

Figure 5
A3AR expression level in PBMCs from RA patients. (a) Newly MTX treated RA patients. Western blots of the four patients at baseline and 10 weeks after MTX treatment are presented. Representative Western blots are shown at the bottom. (b) RA patients undergoing chronic treatment with MTX. Results are expressed as means ± standard error for 30 patients. (c) Double-stained immunofluorecence analysis of PBMCs from RA patients. A3AR expression on the cell surface of the CD4+ T cells was found. (d) Incubation of PBMCs from MTX-treated RA patients for 1 hour in RPMI 1640 supplemented with 10% foetal bovine serum in the presence of with CF101 (10 nmol/l) resulted in decreased expression of A3AR. A3AR, A3 adenosine receptor; AIA, adjuvant-induced arthritis; MTX, methotrexate; PBMC, peripheral blood mononuclear cell; RA, rheumatoid arthritis.
Figure 6

Figure 6
Effect of MTX on A3AR expression level on PBMCs from healthy individuals. (a) PBMCs (2 × 106 cells/ml) from healthy individuals were incubated in RPMI 1640 supplemented with 10% foetal bovine serum and activated with PHA (5 μg/ml) for 24 hours. MTX (1 μmol/l) was added for an additional 27 hours and ADA (1 unit/ml) for the last 3 hours. A3AR expression level was induced by MTX treatment. The introduction of ADA to the culture system reverted this effect and induced downregulation of the receptor level. (b) PBMCs (2 × 106 cells/ml) were incubated for 27 hours with adenosine (25 μmol/l) and ADA (1 unit/ml) was added to the culture system for the last 3 hours. Adenosine induced upregulation of A3AR expression level whereas the addition of ADA decreased the receptor level. (c) PBMCs from healthy individuals were incubated with 10% foetal bovine serum and activated with PHA (5 μg/ml) for 24 hours. MTX (1 μmol/l) was added for an additional 27 hours and CF101 (10 nmol/l), in the presence or absence of MRS1523 (10 nmol/l), was introduced into the culture system for the last 3 hours. CF101 introduction to MTX-treated, PHA-activated PBMCs induced receptor downregulation, and the MRS1523 counteracted this effect. A3AR, A3 adenosine receptor; ADA, adenosine deaminase; MTX, methotrexate; PBMC, peripheral blood mononuclear cell; PHA, phytohemagglutinin; RA, rheumatoid arthritis.

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Об авторе

Александр Забутый

Академик , профессор, доктор сельскохозяйственных наук( Ph.D.Animal science); главный редактор и издатель журнала

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