Using a 92-analyte panel of inflammation-associated markers, we have demonstrated a strong systemic inflammatory profile associated with chronic sarcoidosis. Among the associated inflammatory mediators, serum levels of MIP-1β and TNF-RII were significantly reduced after treatment with infliximab and were associated with greater improvements in FVC and 6MWD. Sarcoidosis patients expressing the highest levels of TNF-α, who had more severe pulmonary and extrapulmonary disease, had the greatest improvement in percent predicted FVC and reduction in MIP-1β and TNF-RII levels after infliximab treatment.
Twenty-nine of 64 analytes quantifiable in the sera of sarcoidosis patients and/or healthy controls were significantly elevated in sarcoidosis patients by at least 1.5-fold over controls. Importantly, these associations were independently observed in both the male and female populations in the gender-stratified analyses. Associated markers include chemokines, neutrophil-associated proteins, acute-phase proteins, coagulation factors, and regulators of metabolism. Many of these associations have not been previously reported for sarcoidosis or have been reported only for associations with expression in airways, but not systemic expression. This strong systemic inflammatory profile prior to infliximab therapy was evident despite the use of corticosteroids and immunosuppressive therapy, and many of these proteins remained elevated after anti-TNF therapy. This suggests that current therapies are still unable to completely suppress the inflammatory response in these patients with sarcoidosis. Why inflammation was substantially reduced in only a small fraction of the infliximab-treated population remains unclear. Potential explanations include the fact that longer active treatment periods are required; there is a limited subset of patients in which TNF-α is a central driver of systemic inflammation, for example, patients with severe extrapulmonary pathology as discussed above; and TNF-α contributes to only a limited component of the overall ongoing inflammation associated with sarcoidosis.
The serum disease profile illustrates an important role for chemokines in the ongoing inflammation associated with sarcoidosis. All chemokines in the panel were significantly associated with sarcoidosis (MCP-1, RANTES, ENA-78, IL-16, etc.). Our findings of these elevated chemokines in sarcoidosis support the following published findings. MCP-1 has been the chemokine most commonly reported to be overexpressed in sarcoidosis, with reports of increased expression in the airways (6
) and systemically in serum (9
). RANTES (10
) and ENA-78 (22
) were reported to have increased expression in the airways in sarcoidosis; we report here an elevation in serum. IL-8, a neutrophil chemoattractant, was reported to be elevated in the airways (6
) and in plasma (4
) and was found to be inversely correlated with FVC (23
) in sarcoidosis patients. We report here that serum levels of IL-8 were modestly inversely correlated with FVC. Our finding of elevated eotaxin in the sera of patients with sarcoidosis is novel.
MIP-1β was reported to be elevated in the bronchoalveolar lavage (BAL) fluid of sarcoidosis patients at all stages of the disease, whereas MIP-1α was elevated only in chronic or progressive disease (5
). Increased serum concentrations of MIP-1α have also been reported by Hashimoto et al. to be associated with sarcoidosis (9
). Although serum levels of MIP-1α were decreased in the sarcoidosis population in the current study, MIP-1α was overexpressed in the TNF-α-high subset of sarcoidosis patients. This subset also was associated with lower percent predicted FVC, consistent with the previous observations of MIP-1α being upregulated only in progressive disease. Treatment with infliximab resulted in a significant decrease in MIP-1β, as well as a trend toward decreases in IL-8 and eotaxin, thus supporting a role for TNF-α in regulating chemokine levels in sarcoidosis.
Interestingly, the elevation of myeloperoxidase and ENRAGE and the inverse correlation of IL-8 with FVC suggests a role for neutrophil activation and phagocytic function in sarcoidosis. It has been previously demonstrated that increased neutrophils in the BAL fluid are associated with a worse prognosis (8
) and that increased neutrophils in BAL fluid correlate with the BAL fluid levels of IL-8 (8
). Based on these results, the utility of monitoring neutrophil-associated proteins in the serum during the course of disease and treatment warrants further investigation.
Sarcoidosis is a disease in which a Th1 phenotype is known to predominate (24
), and IL-12, IL-18, and IFN-γ have been reported to be elevated in sarcoidosis (19
). While IL-18 was elevated in the sarcoidosis patients in our study, IL-12 and IFN-γ were not, as these analytes were below the limit of detection of the assay platform. This platform is designed for large multianalyte profiling, and thus, the detection of the individual analytes is typically not as sensitive as a standard single-analyte assay would be. In addition, these proteins are labile and may be affected by long-term storage and freeze-thaw cycles.
The importance of TNF-α in the development of granulomatous inflammation has been demonstrated in various model systems (24
). However, it is unclear how this translates to sarcoidosis pathology. TNF-α was found at low levels in sarcoidosis patients, but below the quantification limit in almost half of that population. When the sarcoidosis population was stratified into subsets based on relative levels of TNF-α (above versus below the LDD), the subset with higher TNF-α expression presented with more severe pulmonary and extrapulmonary disease and demonstrated a greater increase in percent predicted FVC than the subset with low TNF-α. Patients in the higher TNF-α-expressing group also had a more pronounced inflammatory profile and larger decreases in MIP-1β and TNF-RII serum levels following infliximab treatment. MIP-1β gene expression has previously been demonstrated to decrease in peripheral blood mononuclear cells after treatment with a TNF inhibitor, etanercept, in patients with rheumatoid arthritis (13
). Decreases in TNF-RII following anti-TNF treatment have not previously been reported. These markers may represent a positive pharmacodynamic and response signature to anti-TNF treatment. Overall the results for the TNF-α-stratified subsets support the hypothesis that elevated systemic levels of TNF-α are associated with more severe disease and therefore represent a population potentially more responsive to anti-TNF-α therapy. Therefore, stratification of disease populations based on TNF-α levels may be warranted in future studies of anti-TNF-α therapeutics to assess whether greater clinical efficacy can indeed be attained.
Increased TNF-α levels were observed following infliximab treatment, an apparently paradoxical observation that was nevertheless anticipated. Infliximab stably binds circulating TNF-α, thus stabilizing generally short-lived circulating TNF. It is hypothesized that as new TNF-α is released into circulation, it is likewise bound by infliximab. This would result in an increased pool of nonactive, antibody-bound TNF-α. From previously reported pharmacokinetic analyses (2
), there were substantial levels of free infliximab in circulation at the week 24 endpoint, suggesting that most TNF-α detected was drug bound. Although free versus bound TNF-α has not been formally tested, this is a common observation for anti-TNF therapies in diseases for which they are potently efficacious. Indeed, this was expected and served as a positive control.
In summary, we have demonstrated a 35-analyte disease profile of sarcoidosis that is reflective of a significant degree of systemic inflammation, with many of the inflammation-associated analytes being novel findings. The results reinforce a major role for chemokines in sarcoidosis and provide evidence for granulocyte-mediated inflammation. Overall, infliximab treatment did not result in a substantial reduction of the proteins in the 35-analyte panel; however, MIP-1β and TNF-RII did show decreases following treatment with infliximab.
The current study supports the need for further exploration of anti-TNF therapy for sarcoidosis patients who remain symptomatic despite the use of glucocorticosteroids and/or cytotoxic agents, particularly those expressing the highest serum levels of TNF-α. The impacts of novel therapies with different mechanisms of action on the disease profile will be important in understanding the heterogeneity of the disease. Identification of biomarkers of treatment response may lead to improved management of the unpredictable clinical course of sarcoidosis.