Effects of three anti-TNF-α drugs: Etanercept, infliximab and pirfenidone on release of TNF-α in medium and TNF-α associated with the cell in vitro
Introduction
Tumor necrosis factor-alpha (TNF-α), a proinflammatory cytokine, orchestrates a number of inflammatory responses. The precursor form of TNF-α, transmembrane TNF-α (mTNF), is expressed as a 26-kilodalton type II polypeptide on the cell surface of activated macrophages and lymphocytes as well as other cell types (endothelium). Membrane-bound mTNF is subsequently cleaved by a metalloproteinase, TNF-α converting enzyme (TACE) [1], which releases the secreted soluble form of TNF-α, a 17kD polypeptide. Homotrimerization is required for the biological activities of both forms of TNF-α.
Biological responses to TNF-α are mediated through two structurally distinct receptors: type I and type II. Both receptors are transmembrane glycoproteins with multiple cysteine-rich repeats in the extracellular N-terminal domains. Although their extracellular domains share both structural and functional homology, the intracellular domains are distinct and transduce their signals through both overlapping and distinct pathways. Secreted TNF-α binds to both type 1 receptor (TNFR1) and type II receptor (TNFR2), while the membrane-bound TNF-α binds mainly to type TNFR2 [2], [3], [4], [5]. Like TNF-α, these receptors exist both as a membrane-anchored form, where they mediate the pleiotropic pathophysiological effects of TNF-α, and in a soluble form, where they can bind and neutralize bioactive TNF-α [6], [7], [8].
While the biological effects of soluble TNF-α have been well studied, research continues with a focus on mTNF interactions. Transmembrane mTNF mediates various cytotoxic and inflammatory leukocytic functions via direct cell-cell contact and binding of mTNF to TNFRs on target cells [9], [10], [11]. Recently, the binding of TNFRs to mTNF was found to initiate reverse signaling (i.e., receptor-mediated ligand signal transduction), which can alter the physiology of the mTNF-expressing cell. Binding of anti-TNF-α antibodies or soluble TNFRs to mTNF can phosphorylate an intracellular signaling domain and may cause intracellular calcium changes [11], [12], initiate synthesis and release of various cytokines [11], [13], [14], increase adhesion molecule expression [15], and alter the cellular response to inflammatory stimuli [16].
The central role of TNF-α in the pathogenesis of several chronic inflammatory diseases has led to widespread use of several anti-TNF agents to treat rheumatoid arthritis [17], [18], Crohn's disease [19], psoriasis [20], ankylosing spondylitis [21], [22], and Behcet's disease [23]. Three clinical trials have treated multiple sclerosis with pirfenidone [24], [25], [26].
Three TNF-α binding proteins have been approved by the FDA for human use. Etanercept (Enbrel™) is a recombinant protein of human soluble TNFR2 coupled to Fc portion of human IgG. Infliximab (Remicade™) is a mouse-human chimeric anti-human TNF-α antibody. Adalimumab (Humira™) is a human anti-human TNF-α antibody. All three either act as neutralizing antibodies of secreted TNF-α or absorb TNF-α, thus preventing further interactions with cell surface receptors. However, none of these anti-TNF-α agents have any reported effect on the synthesis of TNF-α. All are administered either intravenously or subcutaneously to be effective.
Pirfenidone [5-methyl-1-phenyl-2-(1H)-pyridone] is a novel, orally-effective anti-fibrotic drug and is currently undergoing Phase III clinical trial for idiopathic pulmonary fibrosis (IPF). It has been demonstrated to inhibit TNF-α synthesis at the translational level [27]. Oku, et al. found that treatment with pirfenidone significantly reduced the LPS-induced elevation of TNF-α, IL-12, and IFN-γ in mice [28]. Also, treatment with pirfenidone protected mice against both endotoxin-induced and Staphylococcus aureus enterotoxin B-induced endotoxic shock as well as the concurrent elevation of serum TNF-α levels [29], [30].
Multiple sclerosis (MS) is characterized by demyelination, progressive axonal loss in the white matter and subsequent neurological deficits. Several lines of evidence associate elevated TNF-α levels with demyelination and the progressive pathogenesis of MS [31], [32], [33], [34]. The importance of TNF-α in secondary progressive multiple sclerosis (SPMS) was further substantiated in two open-label studies [24], [26] and one double-blind, randomized, placebo-control trial [25] where pirfenidone arrested the progression of the disease and stabilized the condition of the patients. In marked contrast, the use of protein-based anti-TNF-α drugs such as infliximab in patients with rapidly progressive MS exacerbated the disease activity and forced the discontinuation of the trial [35]. Furthermore, administration of etanercept for treatment of juvenile rheumatoid arthritis was associated with the onset of MS [36].
We were perplexed as to why two different classes of anti-TNF-α therapies appear to cause such different clinical outcomes in SPMS — exacerbation by protein-based anti-TNF-α drugs versus the stabilization of MS by pirfenidone. With this apparent paradox in mind and given the paucity of data in this area, we chose to study the effects of both drug classes on a relatively simple and well-studied system, LPS-stimulated secretion of TNF-α in the medium and TNF-α associated with the cell in vitro.
Section snippets
Reagents
All reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. Etanercept (Enbrel™) was purchased from a pharmaceutical supplier in 1 cc syringes at 50 mg/ml and was used in both native carrier and dialyzed (in PBS) forms with no differences in efficacy or toxicity between the two. Infliximab (Remicade™) was also purchased from a pharmaceutical supplier in a 100 mg vial containing the lyophilized drug which was reconstituted in sterile PBS prior to use. Heating (95 °C ×
Effects of pirfenidone or anti-TNF-α proteins on cellular cytotoxicity in vitro
The highest concentration of pirfenidone, etanercept or infliximab that did not affect cellular viability was determined by incubating THP-1 cells with or without 1 ng/ml LPS combined with 1 μg/ml to 500 μg/ml pirfenidone, 0.001 μg/ml to 100 μg/ml etanercept, or 0.01 μg/ml to 100 μg/ml infliximab in 96-well plates for 3, 6, 9, or 24 h. Cellular viability was determined by MTS assay (Fig. 1) and trypan blue exclusion (data not shown). In Fig. 1, the results for 6 h incubation are shown as all
Discussion
Chronic inflammation has been found to be at the heart of many diseases, some of which were not initially recognized as having an inflammatory component [40]. As a key early mediator of the inflammatory process, TNF-α has become a major target of numerous pharmaceutical investigations, which have yielded several unique proteins that bind and neutralize TNF-α bioactivity. Because of their relative novelty and recent development, little is known about the long-term consequences of anti-TNF-α
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