Immunopharmacology and Inflammation
Decreased activity of neutrophils in the presence of diferuloylmethane (curcumin) involves protein kinase C inhibition

https://doi.org/10.1016/j.ejphar.2009.03.080Get rights and content

Abstract

Diferuloylmethane (curcumin) has been shown to act beneficially in arthritis, particularly through downregulated expression of proinflammatory cytokines and collagenase as well as through the modulated activities of T lymphocytes and macrophages. In this study its impact on activated neutrophils was investigated both in vitro and in experimental arthritis. Formation of reactive oxygen species in neutrophils was recorded on the basis of luminol- or isoluminol-enhanced chemiluminescence. Phosphorylation of neutrophil protein kinases C alpha and beta II was assessed by Western blotting, using phosphospecific antibodies. Adjuvant arthritis was induced in Lewis rats by heat-killed Mycobacterium butyricum. Diferuloylmethane or methotrexate was administered over a period of 28 days after arthritis induction. Under in vitro conditions, diferuloylmethane (1–100 µM) reduced dose-dependently oxidant formation both at extra- and intracellular level and it effectively reduced protein kinase C activation. Adjuvant arthritis was accompanied by an increased number of neutrophils in blood and by a more pronounced spontaneous as well as PMA (phorbol myristate acetate) stimulated chemiluminescence. Whereas the arthritis-related alterations in neutrophil count and in spontaneous chemiluminescence were not modified by diferuloylmethane, the increased reactivity of neutrophils to PMA was less evident in diferuloylmethane-treated animals. The effects of diferuloylmethane were comparable with those of methotrexate. Diferuloylmethane was found to be a potent inhibitor of neutrophil functions both in vitro and in experimental arthritis. As neutrophils are considered to be cells with the greatest capacity to inflict damage within diseased joints, the observed effects could represent a further mechanism involved in the antirheumatic activity of diferuloylmethane.

Introduction

Diferuloylmethane (curcumin, Fig. 1) is a natural antioxidant derived from rhizomes of Curcuma longa Linn., displaying remarkable antiinflammatory and antiarthritic activities. These beneficial effects have been attributed to the capacity of diferuloylmethane to prevent activation of nuclear factor-kappa B and the subsequent overexpression of proinflammatory mediators — cytokines, adhesion molecules, cyclooxygenase-2, phospholipase A2, myeloperoxidase, collagenase, as well as to its ability to modulate activities of T lymphocytes and macrophages (Huang et al., 2004, Sharma et al., 2005, Funk et al., 2006, Jagetia and Aggarwal, 2007).

Despite the fact that diferuloylmethane is one of the best studied natural compounds, surprisingly minor attention was concentrated on its effects concerning the activity of neutrophils (Srivastava, 1989, Limasset et al., 1993, Jackson et al., 2006). Even though these cells, if activated persistently and excessively, may be toxic to host cells and tissues and may be responsible for the inflammatory tissue damage (Edwards and Hallett, 1997, Vinten-Johansen, 2004). In arthritis, neutrophil-derived oxidants can induce cartilage degradation, depolymerise hyaluronan and decrease its lubricative properties, reduce the protective antioxidant and antiproteinase capacity of synovial fluid and in this way to participate in joint erosion (Edwards and Hallett, 1997, Cross et al., 2006). Downregulation of neutrophil functions (and particularly of their oxidative burst) by diferuloylmethane might thus increase the effectiveness of anti-arthritic therapy and result in disease improvement.

In this study the impact of diferuloylmethane on formation of neutrophil-derived oxidants was studied in vitro and in experimental arthritis. Effects of diferuloylmethane on the concentration of reactive oxygen species produced by neutrophils extra- and intracellularly were separately analysed and the neutrophil protein kinase C activation was examined as an assumed site of diferuloylmethane action. Finally, the effectiveness of diferuloylmethane in vivo was compared with that of methotrexate — a drug widely used in the therapy of patients suffering from rheumatoid arthritis.

Section snippets

Chemicals and solutions

The highly purified diferuloylmethane, luminol, isoluminol, PMA (4β-phorbol-12β-myristate-α13-acetate), Ca2+-ionophore A23187, superoxide dismutase, dextran (average MW 464,000), zymosan (zymosan A from Saccharomyces cerevisiae), luciferase from Photinus pyralis — firefly and d-luciferin sodium salt were from Sigma-Aldrich Chemie (Deisenhofen, Germany), HRP (horseradish peroxidase) and catalase from Merck (Darmstadt, Germany), lymphoprep (density 1.077 g/ml) from Nycomed Pharma AS (Oslo,

Results

Under in vitro conditions, diferuloylmethane (1–100 µM) reduced dose-dependently neutrophil chemiluminescence measured in whole human blood (Table 1). It counteracted stimulation of neutrophils by direct activation of protein kinase C (phorbol myristate acetate), receptor-mediated mechanism (opsonized zymosan) as well as by increased calcium concentration (Ca2+-ionophore A23187).

In isolated neutrophils stimulated with PMA, extra- and intracellular chemiluminescence were recorded separately. As

Discussion

Neutrophil activation is accompanied by the increased formation of reactive oxygen species which may be potentially toxic for surrounding tissues (Edwards and Hallett, 1997, El Benna et al., 2002, Fairhust et al., 2007). This process was found to be reduced by diferuloylmethane, both in vitro and in vivo — after its p.o. administration to arthritic rats. The reduction did not result from the damage of neutrophils by diferuloylmethane, as indicated by no ATP liberation observed in the presence

Acknowledgements

This work was supported by the Slovak Research and Development Agency (APVV-0315-07, APVV-51-017905) and by the Scientific Grant Agency VEGA (2/7019/27).

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