Immune-Regulatory and Molecular Effects of Antidepressants on the Inflamed Human Keratinocyte HaCaT Cell Line

Abstract

Allergic contact dermatitis (ACD) is a T cell-mediated type of skin inflammation resulting from contact hypersensitivity (CHS) to antigens. There is strong comorbidity between ACD and major depression. Keratinocytes release immunomodulatory mediators including pro-inflammatory cytokines and chemokines, which modulate skin inflammation and are crucial cell type for the development of CHS. Our previous studies showed that fluoxetine and desipramine were effective in suppressing CHS in different mouse strains. However, the immune and molecular mechanisms underlying this effect remain to be explored. The aim of the current study was to determine the immune and molecular mechanisms of action of antidepressant drugs engaged in the inhibition of CHS response in the stimulated keratinocyte HaCaT cell line. The results show that LPS, TNF-α/IFN-γ, and DNFB stimulate HaCaT cells to produce large amounts of pro-inflammatory factors including IL-1β, IL-6, CCL2, and CXCL8. HaCaT stimulation was associated with increased expression of ICAM-1, a cell adhesion molecule, and decreased expression of E-cadherin. Imipramine, desipramine, and fluoxetine suppress the production of IL-1β, CCL2, as well as the expression of ICAM-1. LPS and TNF-α/IFN-γ activate p-38 kinase, but antidepressants do not regulate this pathway. LPS decreases E-cadherin protein expression and fluoxetine normalizes these effects. In summary, the antidepressant drugs examined in this study attenuate the stimulated secretion of pro-inflammatory cytokines, chemokines, and modulate adhesion molecule expression by the HaCaT cell line. Therefore, antidepressants may have some clinical efficacy in patients with ACD and patients with comorbid depression and contact allergy.

Keywords: Adhesion molecules; Antidepressant drugs; Contact hypersensitivity; Cytokines; Depression.

Fig. 1

The effect of antidepressants on the level of IL-1β a, IL-6 b, CCL2 c, and CXCL8 d produced by HaCaT cells after stimulation with LPS (left side), TNF-α/IFN-γ (middle), and DNFB (right side). The level of cytokines was measured by ELISA after 24 h of incubation in medium from cell cultures as pg/ml (except IL-1β–lysates, measured in pg/mg protein). Data presented (mean ± SEM) as % control and are derived from three different experiments, where 3 wells of cell culture were used for each group. *p < 0.05 vs. control, #p < 0.05 vs. appropriate stimulated group. Flu, fluoxetine; Des, desipramine; Imi, imipramine; LPS, lipopolysaccharide; TNF/IFN, tumor necrosis factor (TNF)-α/interferon (IFN)-γ; DNFB, 2,4-dinitrofluorobenzene

Fig. 2

The effect of antidepressants on the gene expression of ICAM-1 (left panel; a, c, e) and the protein level of ICAM-1 (right panel; b, d, f) in HaCaT cells after stimulation with LPS, TNF-α/IFN-γ, and DNFB. The expression of ICAM-1 in cell lysates was determined after 4 h of incubation, and the results are presented as the mean fold change (± SEM) relative to the reference gene (GAPDH). ICAM-1 protein level was assessed by ELISA after 24 h incubation; in cell culture lysates, the measurement was done in pg/mg protein, the averaged results are presented as % control (± SEM). The presented data come from three different experiments (for individual types of assays), where for each group, there were 3 wells of cell culture. *p < 0.05 vs. control, #p < 0.05 vs. appropriate stimulated group. Representative fluorescence photomicrographs g showing the effect of TNF-α/IFN-γ and antidepressants: fluoxetine (Flu, 0.5 µM), desipramine (Des, 5 µM), or imipramine (Imi, 1 µM) on ICAM-1 expression in HaCaT cells, incubated for 1 h. The ICAM-1 signal was immunodetected using anti-human, staining with anti-CD54 PE (ICAM-1) antibody, and nuclei were stained using DAPI labeling. Scale bar (20 μm) is located in the bottom right corner of each image. Flu, fluoxetine; Des, desipramine; Imi, imipramine; LPS, lipopolysaccharide; TNF/IFN, tumor necrosis factor (TNF)-α/interferon (IFN)-γ; DNFB, 2,4-dinitrofluorobenzene

Fig. 3

The effect of antidepressants on the gene expression of E-cadherin (left panel; a, c, e) and the protein level of E-cadherin (right panel; b, d, f) in HaCaT cells after stimulation with LPS, TNF-α/IFN-γ, and DNFB. The expression of E-cadherin in cell lysates was determined after 4 h of incubation, and the results are presented as the mean fold change (± SEM) relative to the reference gene (GAPDH). E-cadherin protein level was assessed by ELISA after 24 h of incubation; in cell culture lysates, the measurement was done in pg/mg protein, the averaged results are presented as % of control (± SEM). The presented data come from three independent experiments (for individual types of assays), where for each group, there were 3 wells of cell culture. *p < 0.05 vs. control, #p < 0.05 vs. LPS-stimulated group. Representative fluorescence photomicrographs (g) showing the effect of LPS and antidepressants: fluoxetine (Flu, 0.5 µM), desipramine (Des, 5 µM), or imipramine (Imi, 1 µM) on E-cadherin expression in HaCaT cells, incubated for 24 h. The E-cadherin signal was immunodetected using anti-human, staining with anti-E-cadherin FITC antibody, and nuclei were stained using DAPI labeling. The scale bar (20 μm) is located in the bottom right corner of each image. Flu, fluoxetine; Des, desipramine; Imi, imipramine; LPS, lipopolysaccharide; TNF/IFN, tumor necrosis factor (TNF)-α/interferon (IFN)-γ; DNFB, 2,4-dinitrofluorobenzene

Fig. 4

The effect of antidepressants and stimulation on the level of phosphorylation of proteins: Iκb a, p65 NF-κB , NIK c, and p38 d in HaCaT cells. Protein levels were assessed by Western blotting in samples incubated for 1 h. Histograms show band density normalized to the reference protein, averaged (from 3 different experiments), and presented as % of control ± SEM, with representative immunoblots. *p < 0.05 vs. control group. F, fluoxetine; D, desipramine; I, imipramine; LPS, lipopolysaccharide; TNF/IFN, tumor necrosis factor (TNF)-α/interferon (IFN)-γ; DNFB, 2,4-dinitrofluorobenzene