Abstract
Responders to infliximab therapy have more intermediate monocytes at baseline compared with non-responders http://ow.ly/fMLp300oVrO
To the Editor:
Sarcoidosis is a systemic granulomatous disease of unknown origin that can cause a variety of symptoms, but most often affects the lungs [1]. Because sarcoidosis is self-limiting in the majority of patients, not all patients require therapy [2]. In case of severe sarcoidosis, first-line therapy consists of prednisone, with methotrexate or azathioprine being the most commonly used second-line options [3]. Infliximab, a monoclonal anti-tumour necrosis factor (TNF) drug, is an effective third-line therapeutic for severe sarcoidosis [4, 5].
The exact nature and order of immunological events leading to the formation of sarcoid granulomas remains unknown. Granulomas consist of highly differentiated mononuclear phagocytes (mainly activated macrophages) and lymphocytes [1]. Macrophages of sarcoidosis patients are known to produce excessive amounts of TNF compared with healthy controls [6, 7]. The importance of TNF is underlined by the success of infliximab therapy. As monocytes are the precursors of tissue macrophages, monocytes might be a key player in this process. A promising new therapeutic strategy for sarcoidosis is the combined monocyte and granulocyte apheresis [8]. Although only seven patients were included in a pilot study, the positive effects underline the importance of monocytes in the sarcoidosis disease process.
CD16-expressing monocytes are expanded in various immune-mediated diseases, such as rheumatoid arthritis, diabetes, atherosclerosis, bacterial infections and HIV [9]. High percentages of CD16-expressing monocytes were also found in sarcoidosis patients when compared with controls [10, 11]. CD16-expressing monocytes, and intermediate monocytes in particular, fulfil a pro-inflammatory role by producing TNF as well as other cytokines [12]. We have previously shown that sarcoidosis patients have a significantly higher percentage of the pro-inflammatory intermediate monocytes than healthy controls [13], an observation that has been confirmed recently by Hofer et al. [14]. To date, the few available studies investigating monocytes in sarcoidosis have focused solely on monocyte subsets while any potential relationship between disease activity or response to therapy has not yet been addressed.
In our present study, we analysed whether monocyte subsets in blood can predict the clinical response of sarcoidosis patients to therapy with infliximab (Remicade; Centocor Inc., Malvem, PA, USA). To this end we have studied a selected group of patients with severe sarcoidosis, who were unresponsive to first- and second-line treatment or experienced severe side-effects. These patients received infliximab intravenously according to a standardised protocol starting with 5 mg·kg−1 at weeks 0 and 2 and then every 4 weeks for a period of 26 weeks. This study is part of a prospective, open-label cohort study by Vorselaars et al. [15] evaluating the efficacy of infliximab in sarcoidosis. The institutional review board and the ethics committee approved the study and all patients gave written informed consent.
Peripheral mononuclear blood cells from 38 sarcoidosis patients and 18 healthy controls were stained for CD14, CD16 (eBioscience, San Diego, CA, USA) and HLA-DR (human leukocyte antigen-antigen D related) (BD Biosciences, San Diego, CA, USA) before the start of therapy (baseline) and at 14 and 26 weeks of treatment using flow cytometry. Data were acquired on a FACSCalibur (BD Biosciences) and analysed using FlowJo software (Tree Star, Ashland, OR, USA). We used a gating strategy of FSC/SSC, HLA-DR/SSC and then CD14/CD16 (figure 1a). Monocyte cell counts were determined the day before infusion.
Response to infliximab was defined as a reduction in biomarker levels (soluble interleukin-2 receptor or angiotensin converting enzyme) or maximum standardised uptake value on 18F-fluorodeoxyglucose positron emission tomography ≥40% at week 26 compared with baseline [15]. Using these criteria, 30 patients were classified as responders and eight as non-responders. Flow cytometry data from two responders and one non-responder were missing either at week 14 or 26.
In order to address the question whether monocytes can predict the response to infliximab therapy, we first investigated whether the absolute numbers and the percentages of monocytes and its subsets differed at baseline (figure 1b and c). The absolute number of total monocytes at baseline was increased in responders (0.64×109 cells·L−1) compared with non-responders (0.46×109 cells·L−1, p=0.019) (figure 1b). The absolute number of intermediate monocytes at baseline was also increased in responders (0.023×109 cells·L−1) compared with non-responders (0.011×109 cells·L−1, p=0.011). Interestingly, the percentage of intermediate monocytes at baseline was significantly increased in responders (3.43%) compared with controls (1.82%, p<0.001) (figure 1c). When comparing this group of severe sarcoidosis patients with healthy controls at baseline we found a significant increase in the percentage of intermediate monocytes (3.24% versus 1.82%, p<0.001) (figure 1e), which confirms our data and earlier data [13, 14]. In contrast to what was found by Hofer et al. [14], we found no differences in percentages of intermediate monocytes between males (n=24) and females (n=14, p=0.71).
Furthermore, we studied whether the absolute numbers and percentages of monocytes and its subsets changed during infliximab therapy. Figure 1d shows an example of how the patterns of monocyte subsets of a responder changed during therapy.
Non-responders showed a significant increase in absolute numbers of intermediate monocytes after 26 weeks of therapy (from 0.009×109 cells·L−1 to 0.027×109 cells·L−1; p=0.028). Moreover, non-responders showed significant increases during 26 weeks of therapy in both absolute numbers of total monocytes (p=0.027) and absolute numbers of classical monocytes (p=0.028). Responders showed a significant decrease in non-classical monocytes after 14 and 26 weeks of therapy (from 0.100×109 cells·L−1 to 0.074×109 cells·L−1 to 0.079×109 cells·L−1, p=0.001 and p=0.011, respectively).
Sarcoidosis patients as a total group showed an increase in the percentage of intermediate monocytes from the start of therapy to 14 weeks of therapy (from 3.2% to 4.1%; p=0.010), but fell back to baseline at 26 weeks (figure 1e). Responders also showed an initial slight increase in the percentage of intermediate monocytes after 14 weeks of therapy, but fell back to baseline at 26 weeks. Non-responders showed a significant increase in intermediate monocytes after 26 weeks of therapy (from 2.23% to 3.99%; p=0.018) (figure 1f). Additionally, sarcoidosis patients as a total group showed a significant decrease in the percentage of non-classical monocytes after 14 and 26 weeks of therapy (from 15.4% to 12.6% to 12.3%; p=0.003 and p=0.006, respectively) (figure 1g). Responders also showed a significant decrease in non-classical monocytes after 14 weeks and 26 weeks of therapy (from 15.6% to 13.5% to 12.7%; p=0.012 and p=0.036, respectively) (figure 1h). Non-responders showed a decrease in the percentage of non-classical monocytes as well, but this was not significant.
In conclusion, we show that responders to infliximab therapy have more intermediate monocytes at baseline compared with non-responders. The high level of intermediate monocytes possibly reflects a high inflammatory state, the latter is correlated with a better clinical response to infliximab [15]. The intermediate monocytes of non-responders increase significantly during the initial phase of therapy with infliximab. A similar initial increase of intermediate monocytes following infliximab therapy was seen in patients with Crohn's disease after three infusions [12]. The authors of that work proposed that it could be a compensatory response of the host to TNF depletion, as intermediate monocytes are high TNF producers [12]. Long-term effects were not studied by Nazareth et al. [12].
At baseline, total monocyte numbers (percentage as well as absolute) are decreased in non-responders compared with responders, while during therapy non-responders showed a significant increase of total monocytes. During therapy the non-classical, also pro-inflammatory, monocytes normalise to levels of healthy controls.
Our findings show that the intermediate monocyte population can be used as an indicator for responsiveness to anti-TNF therapy in sarcoidosis. Further investigation of the ratio of monocyte subsets and their function may help us to understand the disease process and predict response to therapy.
Footnotes
Clinical trials: This study is registered at www.trialregister.nl with identifier number NTR3895.
Conflict of interest: None declared.
Support statement: This work was supported by the Innovation Fund of the St. Antonius Hospital (Nieuwegein, The Netherlands).
- Received April 7, 2016.
- Accepted May 2, 2016.
- Copyright ©ERS 2016