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

Cytokines play a major role in both acute and chronic inflammatory processe s, including those produced by sulfur mustard (HD). This study describes re sponses of normal human epidermal keratinocyte (NHEK) cells to 2,2'-dichlor odiethyl sulfide, sulfur mustard (HD), defined by interleukin-l beta (IL-1 beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis facto r-alpha (TNF-alpha) release. A new method for detaching cell to cell adhesi on between keratinocytes has been applied. This method permits the characte rization of endogenous fluid from cellular content that could be applied fo r the development of therapeutic intervention. NHEK (typical average cell d ensity 4.4 x 10(6) cells/mL) were exposed to HD (100 and 300 mu M) in kerat inocyte growth medium (KGM) for 24 h at 37 degrees C in humidified air. Com mercially available enzyme-linked immunosorbent assay (ELISA) kits were use d to measure the cytokine release in NHEK during exposure to 100 and 300 mu M of HD. Exposure to 100 mu M HD increased release of cytokines. IL-1 beta (exposed: 1.41x10(-5)pg/cell +/- 1.60 x 10 (6) pg/cell: control 7.10 x 10 (6) pg cell +/- 1.20 x 10 (6) pg/cell), TNF-alpha (exposed: 1.06 x 10 (6) p g/cell +/- 7.3 x 10 (7) pg/cell; control: 4.04 x 10 (6) +/- 2.80 x 10 (7) p g/cell) and IL-8 (exposed: 3.71 x 10(-5) pg/ cell +/- 3.26 x 10 (6) pg/cell ; control: 2.99 x 10 (6) pg/cell +/- 8.80 x 10 (7) pg/cell) were significan tly enhanced when NHEK cells were detached from culture flasks by non-enzym atic procedures. Cell suspensions of NHEK released low amounts of IL-6 when exposed to 100 mu M for 24 h (exposed: 1.47 x 10(-6) +/- 1.60 x 10 (7) pg/ cell; control: 1.28 x 10 (6) +/- 8.40 x 10 (8) pg/cell). However, cell susp ensions of NHEK increased levels of IL-6 after exposure to 300 mu M HD (4.6 7 x 10 (-5) pg/cell +/- 3.90 x 10 (6) pg/cell; control: 3.99 x 10(-6) pg/ce ll +/- 5.50 x 10 (7) pg/cell). The amount of IL-8 and TNF-alpha present in cell suspensions increased up to 59-fold and fourfold, respectively, above control levels when NHEK cells were exposed to 300 mu M HD. Exposure of NHE K to 300 mu M HD had a highly variable effect on the release of IL-1 beta, where sometimes the secretion of IL-1 beta increased above baseline level a nd other times decreased in cell suspensions. Supernatants were collected f rom cell culture flasks 24 h after exposure of 100 and 300 mu M and signifi cantly increased levels of IL-6 were observed. IL-6 was released in a conce ntration-dependent manner, 3.6-fold up to 8.4-fold, respectively, in supern atant. These proinflammatory mediators IL-1 beta, IL-8, TNF-alpha 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 cutaneou s vesicant injury.