In this study we show that SLE patients receiving single or combined treatment with antimalarial drugs have TNFα serum levels similar to healthy controls, whereas untreated patients and those receiving corticosteroid or immunosuppressive therapies presented increased amounts of this cytokine. These results suggest a very valuable effect of antimalarial treatment by means of the downregulation of in vivo
TNFα levels. Although these pharmacological agents have been widely used as disease-modifying antirheumatic drugs mainly in the treatment of SLE and rheumatoid arthritis [1
], their mechanisms of anti-inflammatory actions have not yet been completely understood. Several in vitro
experiments have demonstrated that quinacrine and related drugs decreased the release of pro-inflammatory cytokines induced by LPS in macrophages [4
]. In mouse models, it has been reported that chloroquine may protect mice from lethal challenge by CpG oligonucleotides and LPS and may decrease serum TNFα and IL-6 in rats injected with sublethal doses of both stimuli [16
]. To the best of our knowledge, however, this is the first report demonstrating an in vivo
association between the use of antimalarial therapy and low levels of serum TNFα, suggesting that the disease-modifying antirheumatic effect of these drugs may be mediated, at least in part, by a strong downregulatory effect on TNFα production.
However, association between antimalarial treatment and TNFα serum levels seems to be influenced by polymorphisms of the genes encoding TNFα and IL-10, indicating that this advantageous connection may only be completely valuable for patients with a specific genotype. Constitutive and induced TNFα and IL-10 production have important interindividual variations that are genetically regulated by SNP at their promoters [8
]. Our data indicate that antimalarial therapy plays a role in the TNFα production of patients who are genetically high TNFα producers. These patients probably have the highest TNFα transcription rates [9
] and consequently the highest serum levels. Although the mechanisms of in vitro
antimalarial-mediated TNFα inhibition [4
] are not yet entirely known, it has been reported that chloroquine inhibited LPS-induced TNFα transcription [6
], interfering with mitogen-activated protein kinase signaling [17
]. Thus, our results suggest that antimalarial agents require a high rate of TNFα transcription to achieve the maximal inhibitory effect. On the other hand, though the relevance of IL-10 was already known in lupus disease, the influence of genetic polymorphisms at the IL-10 promoter on treatment outcome after the use of antimalarials was surprising. Results indicated an involvement of antimalarial treatment in the amount of serum TNFα in SLE patients with a low IL-10 genotype. The limited TNFα downregulatory effect observed in genetically high IL-10 producing patients might be explained by the regulatory feedback mechanism that controls the production of both cytokines, which would lead to a decrease in the TNFα transcription rate in patients producing elevated amounts of IL-10. Supporting this, it has been reported that high IL-10 levels were associated with less effective clearance of Plasmodium falciparum
parasites in patients receiving antimalarial therapy [18
Our data support the idea that the actions of cytokines are profoundly conditioned by the presence of other cytokines, particularly in the case of IL-10 and TNFα, which have opposing roles in systemic inflammatory responses. Thus, on the basis of our previous results, we evaluated the role of the interaction between IL-10 and TNFα genotypes in regulating the response to antimalarial treatment in SLE patients. A strong association was found between carriage of the combined genotype low IL-10/high TNFα and the use of antimalarials for more than three years without the need for any other specific SLE treatment (good responder patients), although the single analysis of the IL-10 genotype did not show significant results. Moreover, the relationship between this combined genotype and treatment outcome was higher than that obtained after single analysis of the gene encoding TNFα, since high IL-10/high TNFα producers were not overrepresented among good responder patients. Taken in conjunction, these results thus indicate that determination of TNFα and IL-10 alleles at the onset of the disease may help identify more suitable candidates for antimalarial treatment and could be used as a genetic predictor of clinical outcome. We would expect SLE patients who are carriers of the pro-inflammatory genotype low IL-10/high TNFα to develop a mild disease presenting a good course under antimalarial therapy. Most of these patients probably developed SLE due to the effect of environmental factors added to their genetically determined high TNFα levels, which could not be modulated by the low production of IL-10. TNFα is a pro-inflammatory cytokine that has been found at elevated levels in the serum of patients suffering SLE and other autoimmune diseases [19
]; it has also been suggested that TNFα genotype influences their susceptibility [10
] and, possibly, their clinical response to treatment. In these patients, the elevated TNFα levels may be involved in diverse pathological mechanisms and, therefore, a clinical benefit is to be expected under a treatment that diminishes TNFα production.
Under the assumption that the elevated TNFα levels found in patients with various chronic inflammatory diseases are deleterious, several anti-TNFα therapies are now available to block the action of TNFα. Actually, TNFα-blockage with antibodies has been of unquestionable clinical benefit to many patients with rheumatoid arthritis, Crohn's disease, psoriasis and, more recently, ankylosing spondilitis [20
]. However, it has been reported that response to this treatment is also influenced by genetic polymorphisms at FcγRIII [27
], HLA-DRB1 [28
], lymphotoxin-α [29
] and TNFα alone [30
] or combined with IL-10 [31
] or lymphotoxin-α [28
]. In fact, TNFα antagonists seem to be more effective in genetically low TNFα producer patients [30
] or with the combined high IL-10/low TNFα genotype [31
]. Several authors have also proposed the use of TNFα lowering agents in the treatment of lupus disease [20
]. Supporting this, an open label study of infliximab in six patients with SLE indicated that TNFα blockage might have a therapeutically beneficial effect, although autoantibodies to double-strained DNA and cardiolipin were increased [34
]. This trend toward an augmented autoantibody production frequently accompanying this treatment was not reported after the use of antimalarials. Given the therapeutical relevance of these results, new studies need to be designed to evaluate the use of these drugs as TNFα downregulators in the treatment of genetically low IL-10/high TNFα producer patients with SLE and other inflammatory diseases who, in addition, are poor responders to TNFα blockage with antibodies.
The limitations of the work were the lack of a prospective longitudinal study analyzing TNFα levels before and after treatment with antimalarials and other SLE therapies and the absence of standardized validated measures of SLE activity.