Anti-rheumatic gold compounds as sublethal modulators of monocytic LPS-induced cytokine secretion
Introduction
Rheumatoid arthritis (RA) is a debilitating and crippling disease that affects 1% of the general population (Lipsky, 1994) and 60–120 million people worldwide (Eisler, 2003). RA is characterized by an autoimmune-mediated proliferation of synovial cells leading to inflammation, pain, and swelling in most major joints (Lipsky, 1994). As the disease progresses over many years, the patient experiences loss of joint function, disability, and premature death (Simon, 2000). Non-steroidal anti-inflammatory agents (NSAIDS) are used palliatively to limit pain and inflammation, but the goal of therapy, not achieved with NSAIDS, is to limit joint damage and suppress the underlying autoimmune dysfunction that causes it (Simon, 2000). Disease-modifying anti-rheumatic drugs (DMARDS) are used to try to achieve this latter goal.
Several different classes of DMARDS have been used to treat RA, including methotrexate, chloroquine, sulphasalazine, D-penicillamine, and gold compounds. All of these drugs have shown some efficacy in treating RA, but all elicit significant and sometimes life-threatening side effects such as bone marrow suppression, ulcerative rashes, and liver or renal dysfunction (Simon, 2000). All also have relatively unknown mechanisms of action on RA. Recent strategies of drug development have focused on neutralizing cytokines that are present in inflamed joints. These cytokines purportedly mediate joint inflammation and destruction in RA, and include TNFα, IL8, and IL6 (Keystone, 2001). The most common of these newer drugs are anti-TNFα drugs, which are available clinically and have enjoyed some success in limiting RA progression. However, these drugs also have severe side effects, including a markedly elevated risk of infection. Furthermore, their focused mechanism of action is increasingly seen as a liability in a disease with a multifactorial etiology. For these reasons, the newest clinical strategies have attempted to combine DMARDS to maximize therapeutic benefits and limit side effects (Keystone, 2001).
Gold compounds have been used for the treatment of RA (chrysotherapy) for at least 100 years (Fricker, 1996, Barrera et al., 1996). Although these drugs have enjoyed much success in limiting joint damage and inducing remission of flares of the disease, the side effects associated with gold compounds have limited their long-term and widespread use (Simon, 2000). Furthermore, the mechanism(s) of action of chrysotherapy remain elusive other than as a general immunomodulatory agent (Fricker, 1996). The two most common gold-based DMARDS used today are auranofin (AF), and gold sodium thiomalate (GSTM), both Au(I) compounds with sulfur-linked organic ligands. The monovalent form of gold appears critical to the clinical efficacy of these drugs, although recent evidence suggests that metabolites of AF and GSTM, including Au(III) salts play a role in the mechanism of action in RA treatment (Fricker, 1996). Identification of the mechanism(s) of action is a critical barrier to the development of additional gold-based drugs and the use of these drugs in combination with newer drugs for RA or perhaps other inflammatory diseases.
Research suggests that gold-based compounds may act on several types of immune cells to achieve their efficacy, such as lymphocytes, neutrophils, or monocytes (Barrera et al., 1996). The ability of gold to modulate the secretion of cytokines from monocytes is one potentially important mechanism by which gold compounds alter the course of RA because monocytes orchestrate many facets of the immune response in rheumatic disease. One prevailing long-term hypothesis is that gold compounds are immunosuppressive, and some research supports the ability of Au(III) and Au(I) compounds to uniformly suppress secretion of cytokines (Wataha et al., 1996) and matrix metalloproteinases (Catrina et al., 2002) from monocytes. Conflicting results and active concentrations among these and other reports undoubtedly stem, at least in part, from differences in variables of study design, including cell type, incubation conditions, exposure times, activation status, and the presence of medium components such as β-mercaptoethanol. Additionally, some suppression of cytokine secretion is likely from relatively non-specific cytotoxic effects (Yang et al., 1995, Wataha et al., 1996).
Newer evidence indicates that gold compounds may selectively increase the number of blood cells producing IL6 and IL10 (Lampa et al., 2002), or differentially suppress secretion of cytokines from activated monocytes either via transcriptional or post-transcriptional mechanisms (Ichibangase et al., 1998, Seitz et al., 2003). However, the differential upregulation of cytokine secretion by AF and GSTM at sublethal concentrations has not been reported. This key result would (1) begin to explain the ability of gold, via modulation of monocytic secretion, to simultaneously limit inflammation in RA yet trigger inflammatory side effects, and (2) suggest opportunities to improve gold-based therapy with combination therapies or new gold-based drugs with different cytokine modulation patterns. Here, we first establish sublethal concentrations of AF, GSTM, and Au(III) as a function of time of exposure to human monocytic cells, then demonstrate that these drugs selectively upregulate the secretion of key inflammatory cytokines from activated monocytic cells at sublethal concentrations.
Section snippets
Gold compounds
Two Au(I) compounds, auranofin (AF, Fisher-ICN) and gold sodium thiomalate (GSTM, Sigma-Aldrich), and a Au(III) compound, (HAuCl4,, 99.8%, Sigma-Aldrich) were used to test our hypothesis that gold differentially modulates secretion of cytokines from activated monocytic cells. AF and GSTM were chosen because they are among the most common gold-based drugs employed clinically to treat RA (Lipsky, 1994, Fricker, 1996, Simon, 2000). Gold chloride was used because it is a soluble form of gold
Cytotoxicity
AF, GSTM, and Au(III) had markedly different dose-response curves as assessed by SDH activity (Fig. 1, Fig. 2, Fig. 3). AF was the most cytotoxic (Fig. 1), exhibiting at least 40–50% SDH suppression at 1 μM, regardless of the exposure time. GSTM (Fig. 2) caused little or no SDH suppression at concentrations as high as 300 μM at 24 and 48 h, and only 25–40% suppression at 72 h. Au(III) (Fig. 3) was intermediate between AF and GSTM in its effect, causing 40–60% SDH suppression at 300 μM. Au(III)
Discussion
The data in the current work suggest that gold compounds are not only immunosuppressive as has been previously suggested (Yang et al., 1995, Wataha et al., 1996, Lampa et al., 2002), but also may upregulate the activation response of monocytes. Among the compounds evaluated in the current study, auranofin (AF) was the most provocative in increasing LPS-induced secretion of key inflammatory cytokines (IL6, IL8) associated with RA (Fig. 4). Upregulation of monocyte activation might play a role in
Acknowledgment
The authors thank the Medical College of Georgia for their support of this work.
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