Effects of pixantrone on immune-cell function in the course of acute rat experimental allergic encephalomyelitis

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Abstract

Pixantrone is an immunesuppressor similar to mitoxantrone but with lower cardiotoxicity. We evaluated the effect of pixantrone on B cells and lymphomononuclear cells in the course of acute EAE. Pixantrone reduced the number of B cells and suppressed myelin basic protein (MBP) specific IgG production. In vitro, pixantrone induced apoptosis of rat B lymphocytes in a way similar to mitoxantrone. In addition, pixantrone inhibited antigen specific and mitogen induced lymphomononuclear cell proliferation, as well as IFN-γ production, during EAE. These findings suggest a similar mechanism of action for pixantrone and mitoxantrone on the effector function of lymphomonocyte B and T cells.

Introduction

Experimental autoimmune encephalomyelitis (EAE), as induced by immunization with central nervous system (CNS) antigens in susceptible animals, is a T cell mediated autoimmune disease of the CNS, widely used as a model for the study of the immunopathological mechanisms of multiple sclerosis (MS) (Bert't Hart and Amor, 2003, Ridge et al., 1985, Wekerle et al., 1994). Various immunosuppressive agents have been found to be effective in preventing and treating EAE, and their effects on T and B cell responses have been elucidated (Levine and Saltzman, 1986, Mustafa et al., 1993).

Mitoxantrone is an immunosuppressive drug effective in acute EAE and recently approved for the treatment of very active MS (Goodin et al., 2003, Hartung et al., 2002). Its prolonged use is, however, limited by its cardiotoxicity (Mather et al., 1987). Mitoxantrone is a potent cytostatic drug and has various distinct immunological effects (Neuhaus et al., 2004). It inhibits the proliferation of macrophages, B and T lymphocytes both in animals (Fidler et al., 1986a, Fidler et al., 1986b) and in MS patients (Gbadamosi et al., 2003). It also has a complex action on the T cell function inhibiting T helper and enhancing T suppressor activities (Fidler et al., 1986b). In particular, unlike other immunesuppressants, mitoxantrone induces apoptosis of B lymphocytes in laeukemic cells (Bellosillo et al., 1998), and in peripheral blood B and T cell during MS (Chan et al., 2005). Moreover, Fidler et al. reported that mitoxantrone decreased secretion of pro-inflammatory cytokines (Fidler et al., 1986b). In contrast, recent ex vivo analysis of the cytokine profile of immune cells obtained from MS patients has shown a more complex pattern of action, as mitoxantrone has revealed a long-lasting decrease of IL-10 expressed in monocytes and of IL-2R-β1 expressed in T cells (Khoury et al., 2002, Gbadamosi et al., 2003). Pixantrone is an antineoplastic drug structurally related to mitoxantrone but with lower cardiac toxicity in comparison to this and other intercalating agents (Krapcho et al., 1994). The mechanism of action of pixantrone is largely unknown but is considered to be the same of mitoxantrone, as both drugs interact with DNA and with topoisomerase II. We recently demonstrated that pixantrone reduces the severity of acute EAE and decreases the relapse rate of chronic EAE in rats and mice (Cavaletti et al., 2004, Gonsette, 2004b). The drug has, therefore, an important potential role in MS treatment and its mechanism of action needs to be further investigated during EAE.

As mitoxantrone mediates a complex immune suppression involving different steps and both B and T lymphocytes, we investigated the effects of pixantrone on immune cell function in acute EAE induced in Lewis rats with the aim to provide further insight into its mechanism of action. We also compared in rat lymphomonuclear (MNC) cells the pro-apoptotic effect of pixantrone in a direct comparison with mitoxantrone.

Section snippets

EAE

Forty inbred female Lewis rats (180–200 g Harlan, Correzzana, Italy) were immunized by subcutaneous inoculation into both hind limb footpads of 50 μg of guinea pig myelin basic protein (Deibler et al., 1972) in complete Freund's adjuvant (3 mg/ml inactivated mycobacterium tuberculosis, Difco). The inoculated rats were randomly assigned to the pixantrone group (PIX, 32.5 mg/kg iv on days 3 and 7 after challenge, n = 20) or the EAE group (sterile saline iv on days 3 and 7, n = 20). Twenty rats were

Antibody response to MBP

The concentration of serum IgG and IgM to MBP was determined at days 10, 14, 23 and 41 p.i. During EAE, we observed increased levels of anti-MBP antibodies from day 10 p.i. until the end of the study (Fig. 1). Antibody production was suppressed by the treatment with pixantrone (p < 0.0001) yielding IgG levels which were comparable to that of CTRL animals at any time of observation (Fig. 1). We could not observe IgM antibodies production.

Spleen cell phenotype

Phenotype analysis of spleen derived mononuclear cells was

Discussion

Mitoxantrone can be considered the most potent treatment of MS (Gonsette, 2004a) and an equally potent immunosuppressive agent targeting transformed cells or proliferating immune cells, such as macrophages, B lymphocytes and T lymphocytes (Fidler et al., 1986a, Fidler et al., 1986b). The immunosuppressant and immunomodulatory properties of mitoxantrone have provided a rationale for its use in active MS (Goodin et al., 2003, Hartung and Gonsette, 1999). Mitoxantrone has a noticeable action on B

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B.M. and T.B. contributed equally to the study.

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