Background/Purpose: Immune-inflammatory cells participate together with endothelial cells and myofibroblasts in the tissue damage that characterizes fibrotic diseases, including systemic sclerosis (SSc) (1). Among immune cells, alternative activated macrophages (M2) are implicated in the pathogenesis of fibrosis through the production of profibrotic molecules (i.e. fibronectin and TGFβ), as observed in lung fibrosis and cancer (1). M2 are characterized by an increased expression of specific markers, such as CD206 (mannose receptor1) and scavenger receptors (CD204 and CD163) and we investigated their presence in the peripheral blood (PB) of SSc patients (pts) (1). Furthermore, since endothelin-1 (ET-1) has been shown to promote the fibrotic process in SSc, we tested possible in vitro effects of ET-1 in inducing the transition of cultured human macrophages into the M2 phenotype (2). 

Methods: Twenty-six randomized female SSc pts (65±10 years), who fulfilled the new EULAR/ACR criteria (3), and ten healthy subjects (HS, 62±10 years) were enrolled into the study after EC approval and Informed Consent signed. Nailfold videocapillaroscopy (NVC) was performed to define the pattern of mycroangiopathy (4). PB from SSc pts and HS was analysed by flow cytometry investigating cells positive for CD204, CD206 and CD14 (monocyte/macrophage marker). Cultured human monocyte cells (THP1, 1×10⁶ cells/ml) were activated to macrophages with phorbol myristate acetate (25ng/ml) and then either untreated or treated with ET-1 (100mM) or IL4 (10ng/ml, as inducer of M2) for 72 hrs in growth medium at 5% of fetal bovine serum. Gene and protein expressions of CD204, CD206 and CD163 were investigated by qRT-PCR and immunocytochemistry. Statistical analysis was performed by Mann-Whitney non-parametric test.

Results: SSc pts showed a significant increased percentage of circulating CD204⁺/CD206⁺cells compared to HS (p<0.05). NVC analysis identified 13 SSc pts with an “early” pattern and 13 SSc pts with an “active/late” pattern of microangiopathy. Of note, the percentage of CD204⁺/CD206⁺cells was significantly increased exclusively in those SSc pts who showed an “early” pattern (p<0.05 vs. HS).  The qRT-PCR showed that ET-1 induced a significant increase in the gene expression of CD204, CD206 and CD163 in cultured macrophages (p<0.05 vs. untreated cells for all M2 markers). In addition, immunocytochemistry showed that ET-1 further increased the protein expression of CD204 and induced the de novo synthesis of CD206 vs. untreated cells. These effects on gene and protein expressions of M2 markers were similar to those induced by treatment with IL4.

Conclusion: Increased percentage of cells with M2 phenotype seems evident in the PB of SSc pts and significantly in those characterized by an “early” NVC pattern of mycroangiopathy. The ability of ET-1 to induce the expression of M2 markers in human macrophages might suggest its possible action in driving the polarization of macrophages into a M2 phenotype.  

References: 1 Wynn TA et al. Nat Med 2012;18:1028-40. 2 Cutolo M et al. J Rheumatol 2015;42:456-63. 3 van der Hoogen F et al. Arthrit Rheum 2013;65:2737-47. 4 Sulli A et al J Rheumatol 2009;36:1235-9.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/increased-circulating-cd204cd206-double-positive-monocytemacrophages-in-systemic-sclerosis-patients-with-early-capillaroscopic-pattern-of-microvascular-damage/