Differential regulation of surface receptor expression, proliferation, and apoptosis in HaCaT cells stimulated with interferon-γ, interleukin-4, tumor necrosis factor-α, or muramyl dipeptide

Abstract

Keratinocytes are routinely subjected to both internal and external stimulation. This study investigates the effects of interferon gamma, interleukin-4, tumor necrosis factor alpha, and the synthetic immunomodulator muramyl dipeptide on the human keratinocyte cell line, HaCaT. Following HaCaT stimulation with cytokines or muramyl dipeptide for different time periods, changes in the expression of different cell surface receptors, cell proliferation, and cell apoptosis were evaluated by flow cytometry, tritiated thymidine uptake, and annexin-V staining, respectively. A significant decrease in the expression of CD49d was found upon treatment with interleukin-4. Interferon gamma and tumor necrosis factor alpha increased the expression of intercellular adhesion molecule 1 and major histocompatibility complex class I, whereas major histocompatibility complex class II and CD1b were only upregulated by interferon gamma. Interferon gamma and tumor necrosis factor alpha had opposite effects regarding CD119 expression, with the former downregulating, while the latter upregulating its expression. Of the stimuli tested, only interferon gamma and tumor necrosis factor alpha significantly inhibited proliferation of HaCaT cells, yet only interferon gamma played a significant role in inducing HaCaT cell apoptosis. Our data demonstrate differential effects of the three tested cytokines on keratinocytes and reveal that the absence of HaCaT cell responses to muramyl dipeptide is associated with undetectable levels of its cytoplasmic receptor, nucleotide-binding oligomerization domain-containing protein 2.

Keywords: CD molecules; apoptosis; cytokines; keratinocytes; muramyl dipeptide; surface receptors.

Figure 1.

Representative flow cytometry histogram plots displaying changes in the expression of CD54, CD95, and CD119 on HaCaT cells 48 h post-stimulation, with cytokines or MDP. Unstim: unstimulated.

Figure 2.

Modulation of surface receptors on HaCaT cells following stimulation with IFN-γ, IL-4, TNF-α, or MDP. Unstimulated cells served as a control. Expression of surface receptors was determined by flow cytometry at 3, 24, 48, and 72 h post-stimulation with 50 ng/mL of each cytokine or 20 µg/mL of MDP. The data shown are representative of three independent experiments (n = 3), values are reported as mean of the total geometric mean fluorescent intensity (MFI) ± SEM. Statistically significant differences were determined by Student’s t-test for unpaired samples (*P < 0.05 vs unstimulated cells).

Figure 3.

Regulation of [3H] thymidine uptake by HaCaT cells following stimulation with IFN-γ, IL-4, TNF-α, or MDP. HaCaT cells were stimulated with (a) 50 ng/mL of each cytokine and 20 µg/mL MDP or (b) 100 ng/mL of each cytokine for 24, 48, and 72 h. Unstimulated cells served as a control. Data are reported as counts per minute (cpm) of tritiated thymidine uptake. The data shown are from three independent experiments. All values are reported as mean ± SEM. Statistically significant differences were determined by Student’s t-test for unpaired samples (*P < 0.05 vs unstimulated cells).

Figure 4.

Cytokine-induced apoptosis in HaCaT cells. HaCaT cells were either left unstimulated or stimulated with IFN-γ, IL-4, or TNF-α at a concentration of 50 ng/mL for 24, 48, and 72 h and apoptosis was determined by annexin V/propidium iodide (PI) staining following the previously indicated stimulation periods. Betulinic acid (BA) was used as a positive control for apoptosis induction. (a) Representative flow cytometry dot plots demonstrating the change in the rate of apoptosis in response to the above-mentioned stimulants. Numbers within the lower right quadrant indicate the percentage of early apoptotic cells, whereas the numbers in the upper right quadrant refer to late apoptotic cells. (b) Changes in the percentage of early (PI⁻/Annexin⁺) and late (PI⁺/Annexin⁺) apoptotic cells. Bars indicate the mean of three independent experiments ± SEM. Statistically significant differences were determined by Student’s t-test for unpaired samples (*P < 0.05 vs unstimulated cells).

Figure 5.

NOD2 expression in HaCaT cells. (a) Total cell lysates from 2 × 10⁶ (lane 1) and 3 × 10⁶ (lane 2) HaCaT cells were immunoprecipitated with NOD2-specific rabbit anti-serum and detected by western blot using the same rabbit NOD2 anti-serum. (b) Membrane in Figure 2(a) was stripped and re-probed with mouse monoclonal NOD2 antibody or its isotype control antibody (Iso). (c) HaCaT total cell lysates were immunoprecipitated with mouse monoclonal NOD2 antibody or its isotype control antibody and detected with NOD2-specific rabbit anti-serum. (d) NOD2 detection in RAW 264.7 total cell lysate (positive control), using mouse monoclonal NOD2 antibody or its Iso.