Increase in circulating cells coexpressing M1 and M2 macrophage surface markers in patients with systemic sclerosis

Alterations in macrophage polarisation are recognised among the possible immune system abnormalities contributing to systemic sclerosis (SSc) pathogenesis.1 Macrophages have been classified as classically (M1) or alternatively (M2) activated, although growing evidence indicates that they may exhibit characteristics shared by more than one of the described phenotypes.2–5 An M2 pre-eminent phenotype has been postulated for SSc monocytes/macrophages.2 The aim of the study was to widen a phenotype characterisation (M1, M2 and mixed M1/M2) of circulating monocytes/macrophages in patients with SSc and healthy subjects (HSs) through flow cytometry analysis. Fifty-eight consecutive patients with SSc (38 limited and 20 diffuse SSc) and 27 age-matched and gender-matched HSs were enrolled after signing informed consent. SSc diagnosis was based on the 2013 American College of Rheumatology/European League Against Rheumatism criteria (online supplementary file 1).6 For flow cytometry analysis, peripheral blood was collected and anti-CD14 and anti-CD45 antibodies were used to identify the monocyte/macrophage lineage; macrophage scavenger receptors (CD204, CD163) and mannose receptor 1 (CD206) were used as M2 phenotype markers; and co-stimulatory molecules (CD80, CD86) and Toll-like receptors (TLR4, TLR2) were used as M1 phenotype markers. CD66b was used to distinguish granulocytes (Miltenyi Biotech, Germany). Flow  c ytometry analysis was performed using the Navios flow cytometer and Kaluza analysis software (Beckman Coulter). A total of  5 × 10 6 cells were evaluated and more than 30 events were detected in the smallest subset investigated, according to the consensus guidelines for minimal residual disease. Results were expressed in percentages over total circulating leu c ocytes, unless otherwise specified. The non-parametric al Mann-Whitney U test was used for statistical analysis and any p value lower than 0.05 was considered statistically significant.  Two initial gating strategies were used to study circulating M2-like monocytes/macrophages, the first gated CD14+cells and the second gated CD204 …