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

Abstract

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.

Keywords: TNF receptors; TNFR1; TNFR2; flow cytometry; gene knockout; receptor expression density.

Figure 1

Flow cytometry gating protocol for identification of cells with TNFR1 and TNFR2 expression. (A) Forward scatter vs. side scatter plot used to identify mononuclear cells and lymphocytes. (B) Dot plot of PE/APC for samples without antibodies (control plot). (C) Use of antibodies TNFR1-PE + TNFR2-APC (experiment plot). (D) Use of antibodies TNFR1-APC + TNFR2-PE (experiment plot). Different colors show 4 fractions of cells co-expressing receptors in different combinations. The distribution of colors on a dot-plot with the phenotypic characteristics of the population shows that all cells are equivalent in size and density.

Figure 2

(A) PCR screening of TNFRSF1A gene deletion. (B) Sample of sequence of TNFRSF1A deletion in ZR-75-01 TNFRSF1A-\-9C9 cells.

Figure 3

Effect of knockout of one of the receptors for TNF on the percentage of expressing cells and the density of receptor expression. %—percentage of cells expressing the corresponding receptor. N—the average number of expressed receptors on the cell surface. The number of replications, n = 5, for each of the experiments.

Figure 4

Dose-dependent effect of TNF cytokine on cell cycle parameters in intact and knockout cell lines. The cells were cultured for 72 h for K-562 and 96 h for ZR-75/1 in the absence and presence of recombinant human TNF at concentrations ranging from 0.01 to 5 ng/mL. The reagent CytoPhase Violet was added to the culture and incubated for 90 min at +37 °C. Analysis was performed using an AttuneNxT flow cytometer. Data are presented as the mean value. The number of replications is n ≥ 3 for each cell line.

Figure 5

Dose-dependent effect of TNF cytokine on cell death parameters in intact and knockout cell lines. The cells were cultured for 72 h for K-562 and 96 h for ZR-75/1 in the absence and presence of recombinant human TNF at concentrations ranging from 0.01 to 5 ng/mL. The cells were stained using Annexin A5 apoptosis detection kit. Data are presented as the mean value. The number of replications is n ≥ 3 for each cell line.