Response of normal human keratinocytes to sulfur mustard (HD): cytokine release using a non-enzymatic detachment procedure

Abstract

Cytokines play a major role in both acute and chronic inflammatory processes, including those produced by sulfur mustard (HD). This study describes responses of normal human epidermal keratinocyte (NHEK) cells to 2,2′-dichlorodiethyl sulfide, sulfur mustard (HD), defined by interleukin-1 beta (IL-1β), interleukin-6 (IL-6), inter-leukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-α) release. A new method for detaching cell to cell adhesion between keratinocytes has been applied. This method permits the characterization of endogenous fluid from cellular content that could be applied for the development of therapeutic intervention. NHEK (typical average cell density 4.4×10⁶ cells/mL) were exposed to HD (100 and 300 μM) in keratinocyte growth medium (KGM) for 24 h at 37°C in humidified air. Commercially available enzyme-linked immunosorbent assay (ELISA) kits were used to measure the cytokine release in NHEK during exposure to 100 and 300 μM of HD. Exposure to 100 μM HD increased release of cytokines. IL-1β (exposed: 1.41×10^-5 pg/ cell±1.60×10^-6 pg/cell: control 7.10×10^-6 pg/ cell±1.20× 10^-6 pg/cell), TNF-α (exposed: 1.06× 10^-5 pg/cell±7.3× 10^-7 pg/cell; control: 4.04×10^-6±2.80×10^-7 pg/cell) and IL-8 (exposed: 3.71×10^-5 pg/ cell±3.26×10^-6 pg/cell; control: 2.99×10^-6 pg/cell±8.80×10^-7 pg/cell) were significantly enhanced when NHEK cells were detached from culture flasks by non-enzymatic procedures. Cell suspensions of NHEK released low amounts of IL-6 when exposed to 100 μM for 24 h (exposed: 1.47×10^-6±1.60×10^-7 pg/cell; control: 1.28×10^-6± 8.40×10^-8 pg/cell). However, cell suspensions of NHEK increased levels of IL-6 after exposure to 300 μM HD (4.67×10^-5 pg/cell±3.90×10^-6 pg/cell; control: 3.99× 10^-6 pg/cell±5.50×10^-7 pg/cell). The amount of IL-8 and TNF-α present in cell suspensions increased up to 59-fold and fourfold, respectively, above control levels when NHEK cells were exposed to 300 μM HD. Exposure of NHEK to 300 μM HD had a highly variable effect on the release of IL-1β, where sometimes the secretion of IL-1β increased above baseline level and other times decreased in cell suspensions. Supernatants were collected from cell culture flasks 24 h after exposure of 100 and 300 μM and significantly increased levels of IL-6 were observed. IL-6 was released in a concentration-dependent manner, 3.6- fold up to 8.4-fold, respectively, in supernatant. These proinflammatory mediators IL-1β, IL-8, TNF-α and IL-6 may play an important role in HD injury. The present findings suggest that cytokine changes detected could be used as potential biomarkers of cutaneous vesicant injury.

Keywords

vesicant agents sulfur mustard ELISA human keratinocytes IL-1β IL-6

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