The anti-inflammatory action of methotrexate is not mediated by lymphocyte apoptosis, but by the suppression of activation and adhesion molecules
Introduction
Methotrexate (MTX) is a potent inhibitor of the enzyme dihydrofolate reductase (DHFR) that leads to the inhibition of de novo purine and pyrimidine synthesis. It was initially developed as a cytostatic agent based on these properties and used for the treatment of oncological diseases in doses of 20–250 mg/kg. Low-dose MTX, which is administered in weekly doses of 0.1–0.3 mg/kg, has been shown to be very effective in the management of arthritic and skin diseases, such as rheumatoid arthritis [1], [2] and psoriasis [3]. Psoriasis is a common T cell-mediated chronic inflammatory skin disease [4], [5]. The skin lesions are characterised by infiltration of T cells into the upper dermis and epidermis and hyperproliferation of keratinocytes. When used for the treatment of psoriasis, MTX was initially thought to act directly against the epidermal hyperproliferation, however, the poor efficacy of locally administered MTX [6], [7], [8] and the effectiveness of agents that primarily target T cells [9], [10] strongly suggest that the anti-proliferative effect of MTX is not responsible for its efficacy in psoriasis.
The inhibition of DHFR is not believed to be the main mechanism of the anti-inflammatory action of MTX, rather, the inhibition of enzymes involved in purine metabolism, which lead to an accumulation of extracellular adenosine, may be of greater importance [11], [12]. Adenosine acts on a variety of cells, but the immunomodulatory effects of adenosine have been best characterised with neutrophils [13]. To date four types of adenoreceptors have been cloned: the A1, A2A, A2B and A3 receptors [13]. Occupancy of neutrophil A1 receptors appears to be pro-inflammatory [14], as it promotes neutrophil adherence to vascular endothelium and chemotaxis, in contrast, A2 receptor engagement inhibits adherence to the endothelium [15] and decreases the generation of reactive oxygen species by neutrophils [14].
It has been suggested that MTX exerts its anti-inflammatory effect by inducing T cell apoptosis and low-dose MTX has been reported to cause apoptosis of mitogen-activated T cells [16]. Resting T cells can take up MTX, convert it into polyglutamated forms that are retained intracellularly, and sensitise them to mitogen-induced apoptosis [17]. There is some evidence that adenosine has cytotoxic and pro-apoptotic properties [18] and in the above system, addition of adenosine increased apoptosis, which was completely reversed by addition of adenosine deaminase. Interestingly, adenosine deaminase also decreased the MTX-induced apoptosis by 10–20%, implicating adenosine, at least in part, in MTX-induced apoptosis of mitogen-activated T cells [17].
Cell adhesion molecules such as vascular cell adhesion molecule (VCAM)-1 and intercellular adhesion molecule (ICAM)-1 are essential for cell-to-cell interactions. ICAM-1 is expressed by T cells, antigen-presenting cells or endothelial cells and binds to the αLβ2 integrin heterodimer leukocyte functional antigen (LFA)-1 expressed by adjacent cells. MTX can reduce inflammatory cell numbers in tissues by affecting the expression of these adhesion molecules [19]. Furthermore, MTX has been shown to reduce inflammatory cell numbers, the expression of monokines and adhesion molecules in the synovial tissue of patients with rheumatoid arthritis [20], and decrease ICAM-1 expression in recipients of rat cardiac allografts [21]. It has also been reported that MTX lowers both the concentration of soluble adhesion molecules [22] and the tissue expression of adhesion molecules such as E-selectin, ICAM-1 and VCAM-1 in patients with bullous pemphigoid [23].
Cutaneous lymphocyte-associated antigen (CLA) is an epitope generated by the modification of P-selectin glycoprotein ligand-1 (PSGL-1) by α(1–3)fucosyl transferase VII (Fuc-TVII) [24]. CLA binds to E-selectin on vascular endothelium in the skin [25] and while it is normally detected on less than 20% of peripheral blood T cells, CLA is expressed by over 80% of the T cells that infiltrate the skin [26], [27]. We have previously demonstrated a correlation between the frequency of CLA+ T cells in the blood of untreated psoriasis patients and their disease severity [28]. Further, we have recently reported a dose-dependent decrease in the expression of CLA by T cells in the blood and skin biopsies of psoriasis patients receiving weekly low-dose MTX treatment and shown that MTX significantly lowered the proportion of T cells expressing CLA, CD103 and ICAM-1 after stimulation with superantigen [29].
Here, we investigate the in vitro effects of low-dose MTX on lymphocytes stimulated with conventional antigens with respect to the induction of apoptosis and the expression of adhesion molecules and explore through which cellular pathways these effects are mediated.
Section snippets
Reagents
MTX and folinic acid were obtained from Wyeth Lederle (Wayne, NJ, USA). Adenosine deaminase (type VI from calf intestinal mucosa), A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), A2 receptor antagonist 3,7-dimethyl-1-propargylxanthine (DMPX) and adenosine were obtained from Sigma (St Louis, MO, USA). The A1 receptor antagonist 1,3-dipropyl-8-phenylxanthine (DPX), A2 antagonist ZM241385, A1 agonist 2-chloro-N-cyclopentyladenosine (CCPA), A2 agonist CGS21680 and staurospaurine
MTX does not induce apoptosis of antigen-stimulated lymphocytes
First, we examined whether the anti-inflammatory effects of MTX are mediated by the induction of apoptosis. MTX caused a significant increase in the number of apoptotic (annexin V-FITC+/PI−) cells in cultures stimulated with the mitogen PHA. However, no such increase was seen in cultures stimulated with either superantigens or conventional antigens (Fig. 1a). Further, a significant decrease in apoptosis was observed in antigen-stimulated cultures in the presence of MTX. Likewise, when cultures
Discussion
This study demonstrates that the anti-inflammatory effects of low-dose MTX are not due to T cell apoptosis, but could be explained, at least partly, by altering T cell activation and adhesion molecule expression. The action of MTX seems to be twofold; first, it acts through folate-dependent pathways to decrease the activation state of antigen-stimulated cells, which decreases activation-induced expression of adhesion molecules by these cells. Second, MTX acts via one or more folate-independent
Acknowledgments
This work was supported by the Icelandic Research Council and the Icelandic Graduate Training Fund. We would like to thank Arna A. Antonsdóttir, Ari Kárason and Ásta Solilja Gudmundsdóttir at deCODE Genetics for help with the TaqMan RT-PCR.
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