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

US Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, Aberdeen Proving Ground, Maryland 21010-5425, USA

R J Schafer

Oak Ridge Institute of Science and Education Research Participation Program

E M Kurt

US Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, Aberdeen Proving Ground, Maryland 21010-5425, USA

C A Broomfield

US Army Medical Research Institute of Chemical Defense, 3100 Ricketts Point Road, Aberdeen Proving Ground, Maryland 21010-5425, USA

A J Carmichael

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-1ß), interleukin-6 (IL-6), inter-leukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-) 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 4.4x10⁶ 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-1ß (exposed: 1.41x10^-5 pg/ cell±1.60x10^-6 pg/cell: control 7.10x10^-6 pg/ cell±1.20x 10^-6 pg/cell), TNF- (exposed: 1.06x 10^-5 pg/cell±7.3x 10^-7 pg/cell; control: 4.04x10^-6±2.80x10^-7 pg/cell) and IL-8 (exposed: 3.71x10^-5 pg/ cell±3.26x10^-6 pg/cell; control: 2.99x10^-6 pg/cell±8.80x10^-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: 1.47x10^-6±1.60x10^-7 pg/cell; control: 1.28x10^-6± 8.40x10^-8 pg/cell). However, cell suspensions of NHEK increased levels of IL-6 after exposure to 300 µM HD (4.67x10^-5 pg/cell±3.90x10^-6 pg/cell; control: 3.99x 10^-6 pg/cell±5.50x10^-7 pg/cell). The amount of IL-8 and TNF- 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-1ß, where sometimes the secretion of IL-1ß 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-1ß, IL-8, TNF- 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.

Key Words: vesicant agents • sulfur mustard • ELISA • human keratinocytes • IL-1ß • IL-6

Human & Experimental Toxicology, Vol. 18, No. 1, 1-11 (1999)
DOI: 10.1177/096032719901800101


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