TNFR1 Absence Is Not Crucial for Different Types of Cell Reaction to TNF: A Study of the TNFR1-Knockout Cell Model.

BACKGROUND: One of the mechanisms regulating the biological activity of tumor necrosis factor (TNF) in cells is the co-expression of TNFR1/TNFR2 receptors. A model with a differential level of receptor expression is required to evaluate the contribution of these mechanisms. AIM: The development of a cellular model to compare the effects of TNF on cells depending on the presence of both receptors and TNFR2 alone. METHODS: TNFR1 absence modifications of ZR-75/1 and K-562 cell lines were obtained by TNFR1 knockout. The presence of deletions was confirmed by Sanger sequencing, and the absence of cell membrane receptor expression was confirmed by flow cytometry. The dose-dependent effect of TNF on intact and knockout cells was comparatively evaluated by the effect on the cell cycle, the type of cell death, and the profile of expressed genes. RESULTS: Knockout of TNFR1 resulted in a redistribution of TNFR2 receptors with an increased proportion of TNFR2+ cells in both lines and a multidirectional change in the density of expression in the lines (increased in K562 and decreased in ZR75/1). The presence of a large number of cells with high TNFR2 density in the absence of TNFR1 in the K562 cells was associated with greater sensitivity to TNF-stimulating doses and increased proliferation but did not result in a significant change in cell death parameters. A twofold increase in TNFR2+ cell distribution in this cell line at a reduced expression density in ZR75/1 cells was associated with a change in sensitivity to low cytokine concentrations in terms of proliferation; an overall increase in cell death, most pronounced at standard stimulating concentrations; and increased expression of the lymphocyte-activation gene groups, host-pathogen interaction, and innate immunity. CONCLUSIONS: The absence of TNFR1 leads to different variants of compensatory redistribution of TNFR2 in cellular models, which affects the type of cell response and the threshold level of sensitivity. The directionality of cytokine action modulation and sensitivity to TNF levels depends not only on the fraction of cells expressing TNFR2 but also on the density of expression.

T-helper cells flexibility: the possibility of reprogramming T cells fate.

Various disciplines cooperate to find novel approaches to cure impaired body functions by repairing, replacing, or regenerating cells, tissues, or organs. The possibility that a stable differentiated cell can reprogram itself opens the door to new therapeutic strategies against a multitude of diseases caused by the loss or dysfunction of essential, irreparable, and specific cells. One approach to cell therapy is to induce reprogramming of adult cells into other functionally active cells. Understanding the factors that cause or contribute to T cell plasticity is not only of clinical importance but also expands the knowledge of the factors that induce cells to differentiate and improves the understanding of normal developmental biology. The present review focuses on the advances in the conversion of peripheral CD4+ T cells, the conditions of their reprogramming, and the methods proposed to control such cell differentiation.

Erythroid Cells as Full Participants in the Tumor Microenvironment.

The tumor microenvironment is an important factor that can determine the success or failure of antitumor therapy. Cells of hematopoietic origin are one of the most important mediators of the tumor-host interaction and, depending on the cell type and functional state, exert pro- or antitumor effects in the tumor microenvironment or in adjacent tissues. Erythroid cells can be full members of the tumor microenvironment and exhibit immunoregulatory properties. Tumor growth is accompanied by the need to obtain growth factors and oxygen, which stimulates the appearance of the foci of extramedullary erythropoiesis. Tumor cells create conditions to maintain the long-term proliferation and viability of erythroid cells. In turn, tumor erythroid cells have a number of mechanisms to suppress the antitumor immune response. This review considers current data on the existence of erythroid cells in the tumor microenvironment, formation of angiogenic clusters, and creation of optimal conditions for tumor growth. Despite being the most important life-support function of the body, erythroid cells support tumor growth and do not work against it. The study of various signaling mechanisms linking tumor growth with the mobilization of erythroid cells and the phenotypic and functional differences between erythroid cells of different origin allows us to identify potential targets for immunotherapy.

Immunoregulatory properties of erythroid nucleated cells induced from CD34+ progenitors from bone marrow.

CD 71+ erythroid nucleated cells have pronounced immunoregulatory properties in normal and pathological conditions. Many populations of cells with immunoregulatory properties are considered candidates for cellular immunotherapy for various pathologies. This study characterized the immunoregulatory properties of CD71+ erythroid cells derived from CD34-positive bone marrow cells under the influence of growth factors that stimulate differentiation into erythroid cells. CD34-negative bone marrow cells were used to isolate CD71+ erythroid nuclear cells. The resulting cells were used to assess the phenotype, determine the mRNA spectrum of the genes responsible for the main pathways and processes of the immune response, and obtain culture supernatants for the analysis of immunoregulatory factors. It was found that CD71+ erythroid cells derived from CD34+ cells carry the main markers of erythroid cells, but differ markedly from natural bone marrow CD71+ erythroid cells. The main differences are in the presence of the CD45+ subpopulation, distribution of terminal differentiation stages, transcriptional profile, secretion of certain cytokines, and immunosuppressive activity. The properties of induced CD71+ erythroid cells are closer to the cells of extramedullary erythropoiesis foci than to natural bone marrow CD71+ erythroid cells. Thus, when cultivating CD71+ erythroid cells for clinical experimental studies, it is necessary to take into account their pronounced immunoregulatory activity.

In Vitro Model of Suppression of the Alloantigen Response by Tolerogenic Dendritic Cells Transfected with Personalized DNA Constructs Encoding HLA Epitopes.

BACKGROUND: A search for efficient graft rejection modulation techniques for the promotion of durable engraftment remains to be a matter of close study all over the world. Despite the variety of immunosuppressive drugs, the schemes currently used show a lack of selectivity and have a number of side effects. Here we investigated an approach for the induction of antigen-specific tolerance in a human "stimulator-responder" model in vitro, using dendritic cells (DCs) transfected with designed DNA constructs encoding the stimulator's major histocompatibility complex (MHC) epitopes. METHODS: The object of the study is peripheral blood mononuclear cells (PBMCs) from 10 healthy donors. To induce antigen-specific tolerance, personalized DNA constructs were created for five responder-stimulator pairs, based on the sequences of donors' and recipients' MHCs. DNA sequencing was performed to select epitopes for incorporation into genetic constructs. A mixed lymphocyte culture assay was used (i) to assess the proliferative response in both directions for all possible stimulator-responder pairs (90 reactions) and (ii) to assess the tolerogenic properties of the generated transfected DCs (5 reactions). RESULTS: A significant increase in the amounts of FoxP3+ CD4+CD25+ cells and in IL-10 production was shown in culture of donor mononuclear cells after co-cultivation with the responder's dendritic cells transfected with donor-specific plasmids. The tolerogenic cultures generated using tolerogenic DCs transfected with MHC epitopes had a significantly greater ability to inhibit the proliferation of autologous MNCs in response to an allogeneic MHC stimulus. CONCLUSIONS: The produced DCs transfected with DNA constructs against HLA stimulating epitopes exhibited tolerogenic properties and may be used to develop antigen-specific tolerance. Thus, we proposed a perspective approach to the induction of antigen-specific tolerance, which should subsequently be studied for use in clinical practice.

The Regulatory-T-Cell Memory Phenotype: What We Know.

In immunology, the discovery of regulatory T (Treg) cells was a major breakthrough. Treg cells play a key role in pregnancy maintenance, in the prevention of autoimmune responses, and in the control of all immune responses, including responses to self cells, cancer, infection, and a transplant. It is currently unclear whether Treg cells are capable of long-term memory of an encounter with an antigen. Although the term "immunological memory" usually means an enhanced ability to protect the body from reinfection, the memory of the suppressive activity of Treg cells helps to avoid the state of generalized immunosuppression that may result from the second activation of the immune system. In this review, we would like to discuss the concept of regulatory memory and in which tissues memory Treg cells can perform their functions.

Autoantibodies to Tumor Necrosis Factor in Patients with Active Pulmonary Tuberculosis.

BACKGROUND: Tumor necrosis factor (TNF) plays an important role in immune responses to the causative agent of tuberculosis, Mycobacterium tuberculosis. Additionally, TNF can also mediate many negative disease manifestations. The aim of this study was to assess the contribution of anti-TNF autoantibodies to the pathogenesis of active pulmonary tuberculosis (TB). METHODS: The levels of anti-TNF autoantibody classes and subclasses were determined by applying enzyme-linked immunosorbent assays (ELISAs). The levels of TNF and of its soluble receptors were also evaluated using commercial ELISA kits. RESULTS: The levels of both types of soluble TNF receptors were lower patients with TB than in healthy donors. Patients with TB had higher titers of immunoglobulin (Ig)G class and IgG3 subclass anti-TNF autoantibodies in comparison with healthy donors. Patients who had a disseminated TB infection had higher TNF level and IgG, IgG1 and IgG3 autoantibody titers compared with patients who had a localized TB infection. CONCLUSIONS: Changes in the titers of anti-TNF autoantibody classes and subclasses were noted in patients with TB, suggesting their possible contribution to the disease pathogenesis of TB.

Dendritic Cells Transfected with MHC Antigenic Determinants of CBA Mice Induce Antigen-Specific Tolerance in C57Bl/6 Mice.

BACKGROUND: Nonspecific immunosuppressive therapy for graft rejection and graft-versus-host disease (GVHD) is often accompanied by severe side effects such as opportunistic infections and cancers. Several approaches have been developed to suppress transplantation reactions using tolerogenic cells, including induction of FoxP3+ Tregs with antigen-loaded dendritic cells (DCs) and induction of CD4+IL-10+ cells with interleukin IL-10-producing DCs. Here, we assessed the effectiveness of both approaches in the suppression of graft rejection and GVHD. METHODS: IL-10-producing DCs were generated by the transfection of DCs with DNA constructs encoding mouse IL-10. Antigen-loaded DCs from C57BL/6 mice were generated by transfection with DNA constructs encoding antigenic determinants from the H2 locus of CBA mice which differ from the homologous antigenic determinants of C57BL/6 mice. RESULTS: We found that both IL-10-producing DCs and antigen-loaded immature DCs could suppress graft rejection and GVHD but through distinct nonspecific and antigen-specific mechanisms, respectively. Discussion. We provide data that the novel approach for DCs antigen loading using DNA constructs encoding distinct homologous determinants derived from major histocompatibility complex genes is effective in antigen-specific suppression of transplantation reactions. Such an approach eliminates the necessity of donor material use and may be useful in immunosuppressive therapy side effects prevention.

Autologous dendritic cells and activated cytotoxic T­cells as combination therapy for breast cancer.

Breast cancer is the most common oncological pathology in women worldwide. Techniques for improving the clinical parameters of patients undergoing combination therapy for breast cancer are currently under development. A type of treatment employing dendritic cells (DCs) and cytotoxic DC­induced antigen­specific T lymphocytes efficiently eliminates residual cancer cells that are the key cause of tumor recurrence and metastasis. In the present study, DCs and activated lymphocytes (treated with IL­12 and IL­18) were isolated from the peripheral blood of patients with breast cancer, using a lysate of tumor tissue as antigen. The patients received the cells as part of adjuvant or neoadjuvant regimens (stage IV disease or progression). Evaluation of immunity was performed at 3 and 6 months after terminating immunotherapy. Evaluation of the disease­free period was performed for 3 years after surgery. The use of antigen­loaded autologous DCs combined with mononuclear cells with increased cytotoxic activity following Th1 polarization reduced the populations of immunosuppressive cells. The results of the present study demonstrated that the investigated cellular immunotherapy for breast cancer is safe, reduces the risk of relapse and metastasis, and improves immunity by reducing the number of regulatory T cells. Therefore, this therapeutic strategy may represent a novel approach to combating distant metastases of breast cancer.

The Effects of Immunosuppressive Factors on Primary Dendritic Cells from C57BL/6 and CBA Mice.

INTRODUCTION: Dendritic cells (DCs) control immune responses by modulating T and B cells towards effector or tolerogenic responses. In this study, we evaluated the effects of different immunosuppressive molecules on the phenotypic and functional characteristics of primary dendritic cells from C57BL/6 and CBA mice. METHODS: DCs were derived from bone marrow cells in the presence of rmGM-CSF and rmIL-4. DCs were then treated with different types of immunosuppressive molecules (rmIL-10, rmTGF-ß, and BAY 11-7082) and cocultured with syngeneic splenocytes. The amount of CD4+CD25hiFoxP3+ Tregs, IL-10 expression, and proliferation were evaluated. RESULTS: Tolerogenic factors were found to have different effects on DCs C57Bl/6 mice. In C57Bl/6 mice, BAY 11-7082 alone had no effect on the expression of DC maturation molecules (CD80, CD86). Transforming growth factor beta (TGF-ß), alone and in combination with BAY 11-7082, reduced the expression of these molecules. Cocultivation of DCs with splenocytes in the presence of TGF-ß and BAY 11-7082 favored regulatory T cell (CD4+CD25hiFoxP3+) differentiation and disfavored differentiation of CD4+ T cells producing IL-10. In CBA mice, we found that rmIL-10 and rmTGF-ß have a weak effect on maturation of DCs and their functional properties to induce Treg cells and IL-10 production. CONCLUSION: These results indicate that TGF-ß and IL-10 have different effects on the phenotypic and functional characteristics of DCs and that the NF-κB inhibitor, BAY 11-7082, has no synergistic effect on these treatments. In mice with an opposite nature of the immune response, the effects of immunoregulatory cytokines (IL-10 and TGF-b) differ on maturation of dendritic cells.

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.

Transfection of bone marrow derived cells with immunoregulatory proteins.

In vitro electroporation gene transfer was first performed in 1982. Today, this technology has become one of the major vehicles for non-viral transfection of cells. All non-viral transfections, such as calcium phosphate precipitation, lipofection, and magnetic transfection, have been shown to achieve a transfection efficiency of up to 70% in commonly used cell lines, but not in primary cells. Here we describe the use of electroporation to transfect primary mouse bone marrow-derived cells, such as macrophages (Mφ) and dendritic cells (DCs) with high efficiencies (45%-72%) and minimal cell death. The transfection efficiencies and cell death varied depending on the culture duration of the DCs and Mφ. Moreover, the electroporation efficiency was increased when conditioning medium was used for culturing the cells. Furthermore, we demonstrated that measuring the plasmid-encoded secreted proteins is a highly sensitive method for determining the transfection efficiency. In summary, electroporation with plasmid vectors is an efficient method for producing DCs and Mφ with transient expression of immunoregulatory proteins.

Production of pure fractions of immunoglobulin G subclass autoantibodies against tumor necrosis factor.

Autoantibodies directed against cytokines are important effector molecules regulating the biological activity of cytokines. There is experimental evidence indicating that autoantibodies belonging to different immunoglobulin G (IgG) subclasses may have different functional activity. The purpose of this work was to develop a protocol for the purification of fractions of IgG subclass antibodies directed against tumor necrosis factor (TNF). We developed a series of steps, including gel filtration, positive and negative affinity chromatography, and ultrafiltration, to achieve this goal. Our protocol purified IgG subclass autoantibodies directed against TNF from a human immunoglobulin preparation. The isolation of these anti-TNF autoantibodies will enable evaluation of the effect of TNF-specific antibodies on TNF biological activity. Our newly developed technique for purifying subclasses of anti-TNF autoantibodies may be important for both basic research on the functional activity of these autoantibodies and for clinical immunology.

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.

Expression density of receptors to IL-1ß in atopic dermatitis.

Interleukin 1 (IL-1 ß) and the system for regulation of its biological effects play an important role in the development and behavior of inflammatory processes in atopic dermatitis. Notably, cells that are actively involved in the pathological process have altered expression of cytokine receptors. However, standard evaluation of cells by flow cytometry measures only the percentage of cells expressing the appropriate marker, which is not enough for a full assessment of these changes. The aim of this study was to investigate changes in the expression of IL-1ß cytokine receptors in patients with atopic dermatitis by both percentage of cells with receptors in various subsets and the absolute number of membrane-bound receptors themselves. It was found that an increase or decrease in the percentage of cells expressing the receptors in subsets of immune cells in patients with atopic dermatitis was not associated with a change in the number of receptors on the cell surface. Moreover, the changes in the percentage of cells and the number of receptors may occur in different directions, as shown for IL-1R2 expression on B cells and IL-1R1 expression for monocytes. Changes in the parameters of IL-1ß receptor expressions are associated with disease severity index SCORAD in atopic dermatitis. These findings underline the importance of studying the density of cytokine receptor expression in the pathology.

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.

Prophylactic Dendritic Cell-Based Vaccines Efficiently Inhibit Metastases in Murine Metastatic Melanoma.

Recent data on the application of dendritic cells (DCs) as anti-tumor vaccines has shown their great potential in therapy and prophylaxis of cancer. Here we report on a comparison of two treatment schemes with DCs that display the models of prophylactic and therapeutic vaccination using three different experimental tumor models: namely, Krebs-2 adenocarcinoma (primary tumor), melanoma (B16, metastatic tumor without a primary node) and Lewis lung carcinoma (LLC, metastatic tumor with a primary node). Dendritic cells generated from bone marrow-derived DC precursors and loaded with lysate of tumor cells or transfected with the complexes of total tumor RNA with cationic liposomes were used for vaccination. Lipofectamine 2000 and liposomes consisting of helper lipid DOPE (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine) and cationic lipid 2D3 (1,26-Bis(1,2-de-O-tetradecyl-rac-glycerol)-7,11,16,20-tetraazahexacosan tetrahydrocloride) were used for RNA transfection. It was shown that DCs loaded with tumor lysate were ineffective in contrast to tumor-derived RNA. Therapeutic vaccination with DCs loaded by lipoplexes RNA/Lipofectamine 2000 was the most efficient for treatment of non-metastatic Krebs-2, where a 1.9-fold tumor growth retardation was observed. Single prophylactic vaccination with DCs loaded by lipoplexes RNA/2D3 was the most efficient to treat highly aggressive metastatic tumors LLC and B16, where 4.7- and 10-fold suppression of the number of lung metastases was observed, respectively. Antimetastatic effect of single prophylactic DC vaccination in metastatic melanoma model was accompanied by the reductions in the levels of Th2-specific cytokines however the change of the levels of Th1/Th2/Th17 master regulators was not found. Failure of double prophylactic vaccination is explained by Th17-response polarization associated with autoimmune and pro-inflammatory reactions. In the case of therapeutic DC vaccine the polarization of Th1-response was found nevertheless the antimetastatic effect was less effective in comparison with prophylactic DC vaccine.

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.

Polymorphisms in the tumor necrosis factor receptor genes affect the expression levels of membrane-bound type I and type II receptors.

The level of TNF receptors on various cells of immune system and its association with the gene polymorphism were investigated. Determining the levels of membrane-bound TNFα receptors on peripheral blood mononuclear cells (PBMCs) was performed by flow cytometry using BD QuantiBRITE calibration particles. Soluble TNF α receptor (sTNFRs) levels were determined by ELISA and genotyping was determined by PCR-RFLP. Homozygous TT individuals at SNP -609G/T TNFRI (rs4149570) showed lower levels of sTNFRI compared to GG genotype carriers. Homozygous carriers of CC genotype at SNP -1207G/C TNFRI (rs4149569) had lower expression densities of membrane-bound TNFRI on intact CD14(+) monocytes compared to individuals with the GC genotype. The frequency differences in the CD3(+) and CD19(+) cells expressing TNFRII in relation to SNP -1709A/T TNFRII (rs652625) in healthy individuals were also determined. The genotype CC in SNP -3609C/T TNFRII (rs590368) was associated with a lower percentage of CD14(+) cells expressing TNFRII compared to individuals with the CT genotype. Patients with rheumatoid arthritis had no significant changes in the frequencies of genotypes. Reduced frequency was identified for the combination TNFRI -609GT + TNFRII -3609CC only. The polymorphisms in genes represent one of cell type-specific mechanisms affecting the expression levels of membrane-bound TNF α receptors and TNF α -mediated signaling.

Quantitative flow cytometric analysis of expression of tumor necrosis factor receptor types I and II on mononuclear cells.

BACKGROUND: Tumor necrosis factor (TNF)-α is an inflammatory cytokine, the biological effects of which are mediated by the interaction with specific membrane-bound receptors. To assess TNF-α receptor (TNFR) expression, it is important to estimate both the number of cells that carry these receptors and the number of receptors per cell, because the cell fate depends on the balance between TNFRI and TNFRII signaling. OBJECTIVE: The aim of the present study was to develop an optimized protocol to estimate the level of expression of membrane-bound TNFRI and TNFRII, using QuantiBRITE PE calibration beads. MATERIALS AND METHODS: The percentage of cells that expressed membrane-bound TNFRI and TNFRII and the mean number of receptors per cell were determined by flow cytometry using PE-labeled antibodies against TNFR. To create a calibration curve and convert cell fluorescence intensity values to absolute numbers of receptors, we used QuantiBRITE PE beads. RESULTS: CD19(+) B lymphocytes had the least percentage of cells expressing TNFRI and the greatest number of receptor molecules per cell, whereas CD3(+) T lymphocytes had the greatest percentage of cells expressing TNFRII and the lowest density of these receptors. We also established that stimulation of peripheral blood mononuclear cells (PBMCs) with the lipopolysaccharide (LPS) significantly increased the number of TNFRI and TNFRII on CD14(+) monocytes. CONCLUSION: Application of the protocol-identified differences in the percentage of cells that expressed TNFRs, as well as the absolute number of receptors per cell, among different subpopulations of PBMCs, and between PBMCs cultured with and without LPS.