Effect of tumor necrosis factor alpha and infliximab on apoptosis of B lymphocytes infected or not with Epstein–Barr virus
Introduction
The incidence of lymphoma is increased in patients with autoimmune diseases such as Sjögren's syndrome, Hashimoto's disease and rheumatoid arthritis (RA) [1], [2], [3], [4], [5], [6]. Most of this increased risk is linked to the persistent activation of autoimmune B lymphocytes, as in Sjögren's syndrome [7], or to the inflammatory activity of the disease, as in RA [8]. Interestingly, the incidence of lymphoma is also increased in patients with non-autoimmune chronic inflammatory diseases such as pyothorax [9] or Crohn's disease (CD) [1].
The increased risk of lymphoma in patients with autoimmune or inflammatory diseases may be also related to immunosuppressive treatment. The two classical drugs that are thought to increase this risk of lymphoma are methotrexate (MTX) and azathioprine. In fact, a few cases of Epstein–Barr virus (EBV)-associated lymphomas have been reported with MTX treatment for autoimmune diseases [10], [11], which regressed after withdrawal of the drug, similar to what is observed in EBV-associated lymphoproliferative disorders in immunodeficient patients [12]. Thus, in RA with lymphoma, MTX could be beneficial, resulting from efficient control of the disease, or deleterious, leading to increased risk of EBV-associated lymphoproliferations. This latter effect seems marginal, since in two large prospective studies, treatment with MTX did not significantly increase the risk of non-Hodgkin's lymphoma with RA [6], [13], although in one of these studies the risk of Hodgkin's lymphoma (usually 10 times less frequent than non-Hodgkin's lymphoma) was increased in RA patients treated with MTX.
Tumor necrosis factor α (TNFα) blockers are a new class of therapeutic agents that are efficient in the treatment of refractory RA, spondylarthropathies and CD. TNFα has a dual role and, depending on the cellular context, can induce cell survival by activating nuclear factor κB (NF-κB) or trigger apoptosis by activating caspases [14]. An increased secretion of TNFα is observed in B lymphocytes upon EBV infection but lymphoblastoid cell lines (LCLs) are resistant to TNFα-induced apoptosis [15] because the viral latent membrane protein 1 (LMP1) protects against it.
Infliximab is a chimeric monoclonal antibody that binds specifically to human TNFα and neutralizes its biologic properties, but some effects of infliximab cannot be explained by the neutralization of soluble TNFα alone [16]. For instance, it induces the rapid suppression of mucosal inflammation in CD and the specific increase of apoptosis in T lymphocytes of the lamina propria in vivo and in vitro. Infliximab also induces apoptosis in synovial monocytes/macrophages in RA [17] and in CD through a caspase-dependent pathway [18].
Results of a few studies suggested an increased incidence of lymphoma in RA patients treated with TNFα blockers [19], [20]. However, to date it has not been possible to discriminate between an increased incidence of lymphoma due to the TNFα blockers themselves and an increased incidence because most severe forms of RA are treated with TNFα blockers. Such increased incidence, which is not established at the present time, could be due either to a defect of immunosurveillance of EBV-infected B cells by T cells or to a direct effect of TNFα blockers on B cells infected or not by EBV. The possibility of an increased risk of lymphoma is one of the major issues concerning the safety of long-term TNFα blocker use.
Only a few data are available concerning the action of TNFα blockers on B cells infected or not with EBV. The purpose of this work was to determine whether TNFα affects apoptosis in EBV-infected B lymphocytes, in Burkitt cell lines infected or not with EBV and in B cells isolated from peripheral blood monoclonal cells (PBMCs), and whether infliximab has a direct effect on apoptosis in these different types of B cells in vitro.
Section snippets
Cell culture
The LCL PRI and the Burkitt lymphoma cell line BL2 and its EBV-infected counterpart BL2.B98.5 were grown at 37 °C in humidified 5% CO2 air in RPMI 1640 medium containing GlutaMAX (GibcoBRL, Life Technologies, Cergy-Pontoise, France) supplemented with 10% decomplemented fetal calf serum (Dutscher, Brumath, France), 100 U/ml penicillin, 10 μg/ml streptomycin (GibcoBRL), 1 mM sodium pyruvate (GibcoBRL), MEM vitamins 100× (GibcoBRL) and 5 μg/ml plasmocin (Cayla InvivoGen, Toulouse, France). Cells were
TNFα, TNFR1 (p55) and TNFR2 (p75) are differentially expressed in EBV-positive B-cell lines
The expressions of TNFα and its receptors were measured in the LCL PRI and BL cell lines infected (BL2.B95.8) or not (BL2) with EBV. The secretion of TNFα was measured by ELISA in cell culture supernatants, and the expression of TNFR1 and TNFR2 was determined by flow cytometry. TNFα secretion was elevated in PRI LCL cells (52 pg/ml) (Fig. 1A). In contrast with BL2 cells, which did not release any detectable amount of TNFα, BL2 B95.8 cells secreted 1 pg/ml of TNFα after 24 h of culture in a new
Discussion
In this study, we demonstrated that neither TNFα nor infliximab had an effect on apoptosis in B cells infected or not with EBV. The increased production of TNFα by EBV-positive cells, particularly LCLs, suggested a possible effect of infliximab on these cells. However, our observation that infliximab had no effect on apoptosis of LCLs, even when cells were sensitized to apoptosis by inhibition of NF-κB suggests that TNFα had no autocrine action in these cells and that infliximab had no
Acknowledgments
This work was supported in part by a grant from Schering-Plough. C.L. was supported by the Fondation pour la recherche médicale (FRM).
References (39)
- et al.
Lymphoma in patients with rheumatoid arthritis: association with the disease state or methotrexate treatment
Semin Arthritis Rheum
(1997) - et al.
Lymphomas in rheumatoid arthritis patients treated with methotrexate: a 3-year prospective study in France
Blood
(2002) - et al.
Reversibility of lymphomas and lymphoproliferative lesions developing under cyclosporin-steroid therapy
Lancet
(1984) - et al.
Apoptotic, non-apoptotic, and anti-apoptotic pathways of tumor necrosis factor signalling
Biochem Pharmacol
(1998) - et al.
Infliximab induces apoptosis in monocytes from patients with chronic active Crohn's disease by using a caspase-dependent pathway
Gastroenterology
(2001) - et al.
Differential roles of STAT1{alpha} and STAT1{beta} in fludarabine-induced cell cycle arrest and apoptosis in human B cells
Blood
(2004) - et al.
NF-kappa B activation by tumor necrosis factor alpha in the Jurkat T cell line is independent of protein kinase A, protein kinase C, and Ca(2+)-regulated kinases
Cytokine
(1991) - et al.
Regulation of human B lymphocyte activation, proliferation, and differentiation
Adv Immunol
(1987) - et al.
The Epstein–Barr virus LMP1 gene product induces A20 zinc finger protein expression by activating nuclear factor kappa B
J Biol Chem
(1992) - et al.
CD40 and its viral mimic, LMP1: similar means to different ends
Cell Signal
(2003)