Co-Expression of Membrane-Bound Tumor Necrosis Factor-Alpha Receptor Types 1 and 2 by Tumor Cell Lines.

INTRODUCTION: Density and co-expression of tumor necrosis factor (TNF) receptors may vary among cell populations. However, the role and potential of these changes remain unclear. This study aimed to determine the density of expression and co-expression of TNFR1/2 and the dose-dependent effect of soluble TNF on these parameters. METHODS: Epithelial-like (HEp-2, K-562, MCF-7, ZR-75/1) and lymphoblast-like (MOLT-4, HL-60, Raji, RPMI-8226, IM-9) cell lines were characterized for co-expression of TNFR1/2 using a modified flow cytometry protocol. The dose-dependent effects of rhTNF on TNF receptor expression in these lines were studied. RESULTS: This study reports a protocol for the simultaneous quantitative evaluation of the of TNF receptor number and co-expression of membrane-bound TNFR1/2. Cells within one tumor cell line were found to differ regarding their expression of type 1 and 2 TNFα receptors; simultaneously, cells with all 4 variants of co-expression may be present in culture. CONCLUSION: We demonstrated a dose-dependent effect of TNF on changes in the expression of TNFR1/2 by the percentage of positive cells and by the number of receptors, which may be used to control TNF-mediated processes in target cells.

Expression Density of Receptors as a Potent Regulator of Cell Function and Property in Health and Pathology.

The expression of cytokine receptors has a crucial role in many cellular processes. Recent studies reported that changes of receptor expression could control the action of mediators on target cells. The initiation of different signaling pathways and, therefore, specific effects on cells, depends on certain components forming the cytokine-receptor complex. These mechanisms control the immune response and affect both the course of diseases (oncological, autoimmune, inflammatory) and the effectiveness of therapy. This review describes the potential of immune mediator receptors to regulate the efficiency of cytokine activity during pathologic processes and ensure the variability of their biological effects. Our aim was to investigate the spectrum of potential roles of changes in mediator receptor expression for main classes of pathologies. For all major types of immune mediators (cytokines, interleukins, chemokines, growth factors, and tumor necrosis factors), it has been shown that changes in their receptor expression are associated with impaired functioning of the organism in chronic diseases.

Expression of TNFα Receptors on Immunocompetent Cells Is Increased in Atopic Dermatitis.

BACKGROUND: Expression levels of cytokine and growth factor receptors have been found to be important in the regulation of their action. Tumor necrosis factor-α (TNFα) is actively involved in inflammation processes in atopic dermatitis (AD), but the role of TNFα membrane receptors (TNFR) and their regulatory function in AD remains unclear. AIM: We aimed to determine the associations of parameters of TNFRα expression on immunocompetent cells with disease severity before and after therapy in AD patients. METHODS: TNFRα expression on T cells, B cells, and monocytes was evaluated by flow cytometry. To determine receptor numbers on the cells, Quantibrite PE beads were used. The content of soluble mediators was evaluated by ELISA. To reveal linear relationships between the index scoring AD (SCORAD) and the studied parameters, multiple linear regression model building was used. RESULTS: TNFR1 and TNFR2 expression in lymphocyte and monocyte populations of AD patients was higher than in healthy individuals (HI). At the same time an increased percentage of positive cells was not associated with high receptor density, and vice versa. Serum content of TNFα, both soluble receptors, the number of TNFR2/T cells, and the percentage of TNFR2+ monocytes were found to be strongly associated with the SCORAD index. CONCLUSION: AD patients had increased TNFR expression on immune cells. Changes in the parameters of TNFRα expression compared to HI were associated with the disease severity index SCORAD.

Levels of TNF, TNF autoantibodies and soluble TNF receptors in patients with bronchial asthma.

OBJECTIVES: The aim of the study was to evaluate the potential contribution made by tumor necrosis factor (TNF) autoantibodies to the pathogenesis of bronchial asthma (BA). METHODS: We used affinity chromatography methods and a magnetic separation procedure to purify human autoantibodies specific to TNF. The autoantibodies were used as a calibration material to determine the absolute content of autoantibodies to TNF using enzyme-linked immunosorbent assay (ELISA). TNF content and levels of soluble receptors to TNF were determined using the ELISA commercial test kits. RESULTS: We demonstrated significant increases in the levels of TNF and soluble TNF receptors in the sera of patients with uncontrolled and controlled BA, as compared with healthy donors. Levels of autoantibodies of the IgG2 and IgG4 subclasses were significantly higher in sera from patients with uncontrolled BA than in healthy donors. Levels of IgG2 autoantibodies were significantly higher in sera from patients with uncontrolled BA than in patients with controlled BA. CONCLUSIONS: BA is associated with changes in the levels of not only TNF and soluble receptors for TNF, but also autoantibodies to TNF. Given the magnitude of the changes in the levels of different subclasses of autoantibodies to TNF, we propose that these autoantibodies might contribute to the pathogenesis of BA.

Optimized flow cytometry protocol for analysis of surface expression of interleukin-1 receptor types I and II.

The biological effects of interleukin (IL)-1 are realized through binding to specific membrane-bound receptors. The efficiency of IL-1 action depends on the number of receptors on the cell. We determined the percentage of cells that express IL-1 receptor type I (IL-1RI) and IL-1 receptor type II (IL-1RII) by flow cytometry using phycoerythrin (PE)-labelled antibodies to the IL-1Rs, and the mean absolute number of membrane-bound IL-1Rs per cell using QuantiBRITE PE calibration beads. We showed that different subpopulations of immunocompetent cells expressed different numbers of molecules of membrane-bound IL-1RI and IL-1RII. We also established that when cells were stimulated with bacterial lipopolysaccharide, there was a significant increase in the number of IL-1RI expressed, and a significant decrease in the mean number of IL-1RII molecules per cell. Determination of the mean number of membrane-bound IL-1R molecules using this protocol enables us to obtain precise and reproducible data that are necessary for full evaluation of expression levels.