Systematic validation of specific phenotypic markers for in vitro polarized human macrophages.

Item does not contain fulltextBACKGROUND: Polarization of macrophages by specific micro-environmental conditions impacts upon their function following subsequent activation. This study aimed to systematically validate robust phenotypic markers for in vitro polarized human macrophages in order to facilitate the study of macrophage subsets in vivo. METHODS: Human peripheral blood monocytes were polarized in vitro with IFN-gamma, IL-4, or IL-10. Similar experiments were performed with TNF, IL-13, dexamethasone, M-CSF and GM-CSF as polarizing stimuli. Phenotypic markers were assessed by flow cytometry and qPCR. RESULTS: IFN-gamma polarized macrophages (MPhi(IFN-gamma)) specifically enhanced membrane expression of CD80 and CD64, IL-4 polarized macrophages (MPhi(IL-4)) mainly upregulated CD200R and CD206, and downregulated CD14 levels, and IL-10 polarized macrophages (MPhi(IL-10)) selectively induced CD163, CD16, and CD32. The expression profiles of the most specific markers were confirmed by qPCR, dose-response experiments, and the use of alternative polarizing factors for each macrophage subset (TNF, IL-13, and dexamethasone, respectively). GM-CSF polarized macrophages (MPhi(GM-CSF)) upregulated CD80 but not CD64 expression, showing a partial phenotypic similarity with MPhi(IFN-gamma), and also upregulated the expression of the alternative activation marker CD206. M-CSF polarized macrophages (MPhi(M-CSF)) not only expressed increased levels of CD163 and CD16, resembling MPhi(IL-10,) but also displayed high levels of CD64. The phenotype of MPhi(M-CSF) could be further modulated by additional polarization with IFN-gamma, IL-4, or IL-10, whereas MPhi(GM-CSF) showed less phenotypic plasticity. CONCLUSION: This study validated CD80 as the most robust phenotypic marker for human MPhi(IFN-gamma), whereas CD200R was upregulated and CD14 was specifically downregulated on MPhi(IL-4). CD163 and CD16 were found to be specific markers for MPhi(IL-10). The GM-CSF/M-CSF differentiation model showed only a partial phenotypic similarity with the IFN-gamma/IL-4/IL-10 induced polarization

Why CCR2 and CCR5 blockade failed and why CCR1 blockade might still be effective in the treatment of rheumatoid arthritis

The aim of this study was to provide more insight into the question as to why blockade of CCR1, CCR2, and CCR5 may have failed in clinical trials in rheumatoid arthritis (RA) patients, using an in vitro monocyte migration system model. Monocytes from healthy donors (HD; n = 8) or from RA patients (for CCR2 and CCR5 antibody n = 8; for CCR1 blockade n = 13) were isolated from peripheral blood and pre-incubated with different concentrations of either anti-CCR1, anti-CCR2, or anti-CCR5 blocking antibodies (or medium or isotype controls). In addition, a small molecule CCR1 antagonist (BX471) was tested. Chemotaxis was induced by CCL2/MCP-1 (CCR2 ligand), CCL5/RANTES (CCR1 and CCR5 ligand), or by a mix of 5 RA synovial fluids (SFs), and cellular responses compared to chemotaxis in the presence of medium alone. Anti-CCR2 antibody treatment blocked CCL2/MCP-1-induced chemotaxis of both HD and RA monocytes compared to isotype control. Similarly, anti-CCR5 antibody treatment blocked CCL5/RANTES-induced chemotaxis of RA monocytes. While neither CCR2 nor CCR5 blocking antibodies were able to inhibit SF-induced monocyte chemotaxis, even when both receptors were blocked simultaneously, both anti-CCR1 antibodies and the CCR1 antagonist were able to inhibit SF-induced monocyte chemotaxis. The RA synovial compartment contains several ligands for CCR1, CCR2, and CCR5 as well as other chemokines and receptors involved in monocyte recruitment to the site of inflammation. The results suggest that CCR2 and CCR5 are not critical for the migration of monocytes towards the synovial compartment in RA. In contrast, blockade of CCR1 may be effective. Conceivably, CCR1 blockade failed in clinical trials, not because CCR1 is not a good target, but because very high levels of receptor occupancy at all times may be needed to inhibit monocyte migration in viv

DELETION OF EITHER CD55 OR CD97 AMELIORATES ARTHRITIS

Tumourgenetics and immunogenetic

Central role of semaphorin 3B in a serum-induced arthritis model and reduced levels in patients with rheumatoid arthritis

Objective: Semaphorin 3B (Sema3B) decreases the migratory and invasive capacities of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) and suppresses expression of matrix metalloproteinases. We undertook this study to examine the role of Sema3B in a mouse model of arthritis and its expression in RA patients. Methods: Clinical responses, histologic features, and FLS function were examined in wild-type (WT) and Sema3B(-/-) mice in a K/BxN serum transfer model of arthritis. Protein and messenger RNA expression of Sema3B in mouse joints and murine FLS, as well as in serum and synovial tissue from patients with arthralgia and patients with RA, was determined using enzyme-linked immunosorbent assay, immunoblotting, quantitative polymerase chain reaction, and RNA sequencing. FLS migration was determined using a wound closure assay. Results: The clinical severity of serum-induced arthritis was significantly higher in Sema3B(-/-) mice compared to WT mice. This was associated with increased expression of inflammatory mediators and increased migratory capacity of murine FLS. Administration of recombinant mouse Sema3B reduced the clinical severity of serum-induced arthritis and the expression of inflammatory mediators. Sema3B expression was significantly lower in the synovial tissue and serum of patients with established RA compared to patients with arthralgia. Serum Sema3B levels were elevated in patients with arthralgia that later progressed to RA, but not in those who did not develop RA; however, these levels drastically decreased 1 and 2 years after RA development. Conclusion: Sema3B expression plays a protective role in a mouse model of arthritis. In RA patients, expression levels of Sema3B in the serum depend on the disease stage, suggesting different regulatory roles in disease onset and progression.</p