Reduced expression of monocyte CD200R is associated with enhanced proinflammatory cytokine production in sarcoidosis

www.nature.com/scientificreports OPEN received: 11 July 2016 accepted: 11 November 2016 Published: 08 December 2016 Reduced expression of monocyte CD200R is associated with enhanced proinflammatory cytokine production in sarcoidosis Simon D. Fraser1, Laura R. Sadofsky1, Paul M. Kaye2 & Simon P. Hart1 In sarcoidosis, the proinflammatory cytokines interferon gamma, tumour necrosis factor and interleukin-6 are released by monocyte-derived macrophages and lymphocytes in the lungs and other affected tissues. Regulatory receptors expressed on monocytes and macrophages act to suppress cytokine production, and reduced expression of regulatory receptors may thus promote tissue inflammation. The aim of this study was to characterise the role of regulatory receptors on blood monocytes in patients with sarcoidosis. Cytokine release in response to stimulation of whole blood was measured in healthy controls and Caucasian non-smoking patients with sarcoidosis who were not taking disease modifying therapy. Expression of the regulatory molecules IL-10R, SIRP-α/β, CD47, CD200R, and CD200L was measured by flow cytometry, and functional activity was assessed using blocking antibodies. Stimulated whole blood and monocytes from patients with sarcoidosis produced more TNF and IL-6 compared with healthy controls. 52.9% of sarcoidosis patients had monocytes characterised by low expression of CD200R, compared with 11.7% of controls (p < 0.0001). Patients with low monocyte CD200R expression produced higher levels of proinflammatory cytokines. In functional studies, blocking the CD200 axis increased production of TNF and IL-6. Reduced expression of CD200R on monocytes may be a mechanism contributing to monocyte and macrophage hyperactivation in sarcoidosis. Sarcoidosis is characterised by increased inflammatory activity within tissue granulomata, with accumulation of activated lymphocytes and monocyte-derived macrophages (epithelioid macrophages) and local release of proinflammatory cytokines1–3. Lung macrophages, derived from blood monocytes4, are potent producers of TNF and IL-65–7 which contribute to the formation of sarcoid granulomata8. Regulation of inflammatory responses is vital to initiate resolution and prevent excessive tissue damage9. Abnormalities of regulatory pathways that normally act to dampen inflammation could explain the hyper-active immunological state seen in sarcoidosis. Interleukin-10 (IL-10) is the archetypal regulatory cytokine involved in control of Th1 immune activity. IL-10 is produced primarily by monocytes and regulatory T lymphocytes, and acts through its receptor IL-10R on T cells, monocytes, and macrophages10. Cranshaw et al. reported that sarcoidosis blood monocytes produced smaller amounts of IL-10 than controls and were less able to suppress T cell proliferation11. Monocytes and macrophages also express signal-regulatory protein-alpha (SIRP-α) which binds to ubiquitously expressed CD47 and acts as an anti-phagocytic signal12, and CD200R13, which binds its cognate ligand CD200L on many cell types and leads to reduced mitogen-activated protein kinase signalling through recruitment of Ras GTPase activating protein14. The CD200R/CD200L axis is vital in maintaining immune homeostasis in the lungs of mice14,15, and reduced CD200R signalling has been implicated in the pathology of joint inflammation16, neurodegeneration17, and cancer18. The aim of the present study was to assess the role of regulatory receptors in modulating monocyte cytokine production in sarcoidosis. A whole blood assay was chosen for monocyte stimulation to minimise ex vivo perturbation of monocytes. Monocytes are shown to be a source of TNF and IL-6 within stimulated whole blood assays, 1 Department of Academic Respiratory Medicine, Centre for Cardiovascular and Metabolic Research, Hull York Medical School, Castle Hill Hospital, Cottingham, HU16 5JQ, UK. 2Centre for Immunology and Infection, Department of Biology and Hull York Medical School, University of York, Wentworth Way, York, YO10 5DD, UK. Correspondence and requests for materials should be addressed to S.P.H. (email: ) Scientific Reports | 6:38689 | DOI: 10.1038/srep38689 1 www.nature.com/scientificreports/ Category Healthy Number 31 Sarcoidosis 30 Age (years) 41 (20–72) 51 (28–73) Male/female 13/18 16/14 — 33 Scadding chest x-ray stage (%) 0 1 7 2 43 3 17 Serum ACE activity (reference range 8–65 U/L) — 84.5 (25–356) C-reactive protein (reference range 0–8 mg/L) — 3.8 (0.5–64) Plasma viscosity (reference range 1.5–1.72 m.Pas.s) — 1.68 (1.49–1.93) Table 1. Demographics and clinical data for healthy subjects and patients with sarcoidosis. Data are presented as median (range). and monocytes from sarcoidosis patients produced these cytokines to a greater extent than healthy volunteers. Patients with sarcoidosis more commonly had monocytes expressing low levels of CD200R, whereas other regulatory receptors (IL-10R, SIRP-α) were expressed at normal levels. Finally, blockade of CD200R or CD200L led to increased production of TNF and IL-6. Collectively, the data argue that reduced expression of CD200R is an important mechanism underlying monocyte/macrophage hyper-responsiveness in sarcoidosis. Results Patients with sarcoidosis display T lymphocytopenia. The demographics and clinical details of study participants are shown in Table 1. All subjects were Caucasians. Patients with sarcoidosis were all non-smokers and were not taking corticosteroids or other disease modifying therapies. Twelve subjects with sarcoidosis (40%) had a Scadding stage 0 or 1 chest X-ray (i.e. without visible lung changes) and 18 (60%) had a stage 2 or 3 chest X-ray. All subjects had CT scan evidence of lung parenchymal abnormalities or mediastinal lymph node enlargement. Immunophenotyping of PBMCs showed that patients with sarcoidosis exhibited a general T lymphocytopenia, in keeping with previous reports19 (Supplementary Table S3 and Supplementary Fig. S1). Sarcoidosis monocytes produce more TNF and IL-6. A whole blood assay was used for ex vivo stimulation studies. Significantly higher concentrations of secreted TNF and IL-6 were found in stimulated whole blood from patients with sarcoidosis compared with healthy controls (Fig. 1). IFNγ and IL-10 were not significantly different between sarcoidosis patients and healthy controls (Fig. 1). When the kinetics of cytokine production in PHA-stimulated whole blood were measured, IFNγ and IL-10 were produced with kinetics commensurate with T lymphocyte activation, whereas TNF production was rapid, peaking at 3–6 hours and declining thereafter (Supplementary Fig. S2). Similar kinetics have been observed by others for monocyte-derived TNF20,21. PHA is a T cell mitogen22 and it also stimulates monocytes by cross-linking Toll-like receptors23. To further explore the relative contribution of T lymphocytes and monocytes to the enhanced cytokine release observed in sarcoidosis, whole blood was st (...truncated)