Co-Expression Profile of TNF Membrane-Bound Receptors Type 1 and 2 in Rheumatoid Arthritis on Immunocompetent Cells Subsets.

INTRODUCTION: Tumor necrosis factor (TNFα) is an important proinflammatory cytokine in rheumatoid arthritis (RA) immune processes. However, TNFα activity and functions may be regulated by soluble receptors, which act as decoys, and by number, density, and co-expression of its membrane-bound receptors type 1 and 2 (TNFR1 and TNFR2). The aim of this study was to reveal associations between TNFR1/2 co-expression profile parameters and RA disease activity indicators. METHODS: PBMC were analyzed from 46 healthy donors and 64 patients with RA using flow cytometry. Patients were divided according to the disease activity score (DAS) 28 index into groups with high (n = 22, 34.4%), moderate (n = 30, 46.9%), and low (n = 12, 18.8%) disease activity. Co-expression of TNFR1 and TNFR2 was studied by evaluating the percentage of cells, with different receptors, and by counting the number of receptors of each type per cell, using QuantiBritePE beads. Associations between disease severity and activity indicators and parameters of TNFα receptor expression in subpopulations of immune cells were studied. RESULTS: T cell subsets from RA patients were characterized by co-expression of TNFR1 and TNFR2, and were found to differ significantly compared with healthy donors. Memory cells both among T helper cells and cytotoxic T cells demonstrated the most significant differences in TNFR-expression profile. Multivariable logistic regression revealed model to identified RA patients from healthy individual based on the TNFR1/2 co-expression parameters. CONCLUSION: The profile of TNFR1\2 co-expression differs in RA comparing with health. Proportion of TNFR1+TNFR2- cells increased significantly among memory T helper cells and activated cytotoxic T cells, and decreased significantly among naïve cytotoxic T cells and T regulatory cells as compared with health. The parameters of TNFR1\2 co-expression in RA are associated with clinical and laboratory indicators of disease activity.

Expression of TNFα membrane-bound receptors in the peripheral blood mononuclear cells (PMBC) in rheumatoid arthritis patients.

OBJECTIVE: To investigate the expression of TNFα membrane-bound receptors: the percentage of cells expressing these receptors and the number of molecules expressed on different immune cell subsets, and to evaluate serum concentrations of soluble TNFα and its receptors (sTNFRI and sTNFRII) in patients with rheumatoid arthritis in acute stage and after response to treatment compared to healthy donors. METHODS: The objects of the study are peripheral blood mononuclear cells (PBMC) of healthy donors (n=150) and RA patients (n=40) subjected to hospital treatment with either biological agents (Rituximab) or glucocorticosteroids (methylprednisolone). To determine PBMC phenotype antibodies anti-hCD3-APC, anti-hCD19 PECy7, anti-hCD14 FITC (eBioscience), as well as anti-hTNFRI-PE and anti-hTNFRII-PE (R&D Systems) were used. To determine receptor number on the cells Quantibrite PE Beads (BD) were used. RESULTS: Cells obtained from patients who responded to therapy and achieved disease remission exhibited either an increase in the percentage of TNFRI+ cells or elevated expression density of this receptor type. CONCLUSION: Subsets of immunocompetent cells from RA patients show variation in the percentage of membrane-bound receptor positive cells and receptor expression density, which influences the development and progression of the pathological processes in RA. Response to therapy and achievement of disease remission are associated with an increase of TNFRI expression.

Differences of IL-1ß Receptors Expression by Immunocompetent Cells Subsets in Rheumatoid Arthritis.

IL-1ß is involved in the induction and maintenance of chronic inflammation in rheumatoid arthritis (RA). Its activity is regulated and induced by soluble and membrane-bound receptors, respectively. The effectiveness of the cytokine depends not only on the percentage of receptor-positive cells in an immunocompetent subset but also on the density of receptor expression. The objective of this study was to investigate the expression of IL-1ß membrane-bound receptors (IL-1R1 and IL-1R2) in terms of the percentage of receptor-positive cells and the number of receptors per cell in different subsets of immune cells in RA patients before and after a course of basic (excluding anticytokine) therapy and in healthy individuals. The resulting data indicate differences in the expression of IL-1ß receptors among T cells, B cells, and monocytes in healthy volunteers and in rheumatoid arthritis patients. The importance of determining both the relative percentage of cells expressing receptors to immunomodulatory cytokines and the number of membrane-bound receptors per cell is highlighted by evidence of unidirectional or multidirectional changing of these parameters according to cell subset and health status.

Parameters of TNF receptor co-expression in allergic and autoimmune processes: Differences and diagnostic significance.

The authors used a method quantitative estimation density of TNFR1/TNFR2 on cells by flow cytometry with calibration particles, which allowed them to estimate the absolute number of receptors on cells regardless of the type of flow cytometer. The TNF receptor expression parameters were used to determine their association with the fact of disease and to build diagnostic models. The proposed methodological approach using a combination of flow cytometry and mathematical modeling techniques represents a promising direction for testing the diagnostic and prognostic significance of the studied biomarkers. The multifactorial regression analysis constructed on the basis of this approach made it possible to refine and supplement diagnostic schemes for determining the probability of rheumatoid arthritis and bronchial asthma in patients.

The Influence of Severity and Disease Duration on TNF Receptors' Redistribution in Asthma and Rheumatoid Arthritis.

One of the mechanisms of cellular dysfunction during the chronization of immune-system-mediated inflammatory diseases is a change in the profile of expression and co-expression of receptors on cells. The aim of this study was to compare patterns of redistribution of TNF receptors (TNFRs) among patients with different durations of rheumatoid arthritis (RA) or asthma. Subgroup analysis was performed on RA (n = 41) and asthma (n = 22) patients with disease duration<10 years and >10 years and on 30 comparable healthy individuals. The co-expression profile of TNFR1 and TNFR2 was assessed in T cells, B cells, monocytes, regulatory T cells, T-helper subsets, and cytotoxic T-lymphocyte subsets. Percentages of cells with different co-expression combinations and receptor density per cell were estimated. Longer disease duration was significantly associated with a redistribution of receptors in immunocompetent cell subsets with an increase in the expression of TNFR1 in asthma but did not correlate with significant unidirectional changes in receptor expression in RA. In asthma, a higher proportion of cells with a certain type of TNF receptor (as compared with the healthy group) was correlated with a simultaneous greater density of this receptor type. In RA, an inverse correlation was observed (compensatory lower receptor density). Mechanisms of long-term changes in the expression of TNF receptors differ significantly between the diseases of autoimmune and allergic etiology. The formation of irreversible morphostructural alterations was strongly correlated with changes in the expression of TNFR1 in asthma and with changes in the expression of TNFR2 in RA.

T-regulatory cells from patients with rheumatoid arthritis retain suppressor functions in vitro.

Rheumatoid arthritis (RA) is a chronic disease of connective tissue caused by intolerance to self-antigens. Regulatory T cells (Tregs) are key players in maintaining autotolerance through a variety of suppressor mechanisms. RA is generally believed to develop due to disorders in Tregs; however, there is no consensus on this issue. Thus, the present study focused on phenotypical analysis of Treg cells and their ability to suppress CD4+ and CD8+ cell proliferation. The present study used peripheral blood samples from 21 patients with RA and 22 healthy donors. The CD25+FoxP3+ subpopulation of Tregs was analyzed using flow cytometry to evaluate the expression of CTLA-4, PD-L1, HLA-DR, CCR4, CD86 and RORyt. Tregs suppressor activity was calculated in terms of suppression of the proliferation of CD4+ and CD8+ lymphocytes in vitro. Suppressor activity of the total Treg population did not differ between patients with RA and healthy donors. However, the patients had elevated CD25loFoxP3+ levels and lower CD25hiFoxP3+ levels; in addition, they had more activated Tregs expressing PD-L1, HLA-DR, CCR4 and CD86. The surface expression of CTLA-4 was below the reference level. The patients also had transitional FoxP3+RORyt+ cells and elevated CD4+RORyt+ levels, which were highly correlated with disease activity. These results show that in RA, Treg cells are activated and have an immunosuppressive activity. However, it is the transitional FoxP3+RORyt+ cells and increased CD4+RORyt+ percentages in peripheral blood that appear to be associated with the pathological conversion of some Treg cells into Th-17. This process appears to be key in RA pathogenesis.

Maturation and cytokine production potential of dendritic cells isolated from rheumatoid arthritis patients peripheral blood and induced in vitro.

BACKGROUND: Since dendritic cells (DC) are involved in the development of autoimmune inflammation, researchers consider DC both as target cells for specific therapy of rheumatoid arthritis (RA) and as candidate cells for the development of cell-based methods to treat autoimmune diseases. The development of treatment strategies requires comprehensive research into the quantitative and qualitative characteristics of DC subtypes both ex vivo from RA patients and in vitro, to determine the possibility of inducing functionally mature DC in RA. OBJECTIVE: To study the phenotypic and functional properties of myeloid (mDC) and plasmacytoid (pDC) DC isolated from the peripheral blood of patients with RA and induced in vitro. MATERIALS AND METHODS: Blood samples were obtained from RA patients and healthy donors. Immature DC in the whole blood and in vitro induced DC were characterized by the positive expression of CD80, CD83, CCR7, IL-10, IL-4, IL-12 and IFN-α. R848 and lipopolysaccharide were used to determine DC maturation ability. From PBMCs of RA patients and health donors DCs with myeloid (imDC) and plasmacytoid (ipDC) phenotype were induced. RESULTS: The relative count of mDC in the peripheral blood between studied groups did not differ. pDC count was significantly lower for RA patients. DC from RA patients were characterized by low expression levels of CD80 and CD83 on both populations cells and high expression of CCR7 only on pDC. An increase in pDC producing IL-12 and IFN-α and a decrease in mDC and pDC producing IL-4 and IL-10 were shown in RA. imDC and ipDC obtained from RA patients according to their phenotype and cytokine profile did not differ from those obtained from healthy donors. CONCLUSIONS: There is an imbalance between subpopulations of DC in the peripheral blood of RA patients. DC of RA patients are less mature. The data suggest the involvement of DC in RA pathogenesis and confirm DC participation in balance shift towards Th1-type immune responses. At the same time, in vitro induced RA DC are phenotypically and functionally competent.