Response of normal human keratinocytes to sulfur mustard (HD): cytokine release using a non-enzymatic detachment procedure
Response of normal human keratinocytes to sulfur mustard (HD): cytokine release using a non-enzymatic detachment procedure
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DOI：
10.1191/096032799678839329
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发表时间：
1999-01-01
影响因子：
2.8
通讯作者：
Carmichael, AJ
中科院分区：
文献类型：
--
作者：
Arroyo, CM;Schafer, RJ;Carmichael, AJ
细胞因子在急性和慢性炎症过程中起主要作用，包括硫芥（HD）产生的细胞因子。本研究描述了正常人表皮角质形成细胞（NHEK）对2，2 '-二氯二乙基硫醚、硫芥（HD）的反应，其通过白细胞介素-1 β（IL-1 β）、白细胞介素-6（IL-6）、白细胞介素-8（IL-8）和肿瘤坏死因子-α（TNF-α）的释放来定义。一种新的方法分离角质形成细胞之间的细胞粘附已被应用。该方法允许表征来自细胞内容物的内源性流体，其可应用于治疗干预的开发。将NHEK（典型的平均细胞密度4.4 × 10（6）细胞/mL）暴露于在角质形成细胞生长培养基（KGM）中的HD（100和300 μ M）中，在37 ℃的潮湿空气中24小时。使用市售酶联免疫吸附测定（ELISA）试剂盒测量暴露于100和300 μ M HD期间NHEK中的细胞因子释放。暴露于100 μ M HD增加细胞因子的释放。IL-1 β（暴露：1.41 × 10（-5）pg/细胞+/- 1.60 × 10（6）pg/细胞：对照7.10 × 10（6）pg/细胞+/- 1.20 × 10（6）pg/细胞），TNF-α（暴露：1.06 × 10（6）pg/细胞+/- 7.3 × 10（7）pg/细胞;对照：4.04 x 10（6）+/- 2.80 x 10（7）pg/细胞）和IL-8（暴露：3.71 x 10（-5）pg/细胞+/- 3.26 x 10（6）pg/细胞;对照：2.99 × 10（6）pg/细胞+/- 8.80 × 10（7）pg/细胞）显著增强。当暴露于100 μ M 24小时时，NHEK的细胞悬液释放少量的IL-6（暴露：1.47 x 10（-6）+/- 1.60 x 10（7）pg/细胞;对照：1.28 x 10（6）+/- 8.40 x 10（8）pg/细胞）。然而，在暴露于300 μ M HD后，NHEK的细胞悬浮液增加了IL-6的水平（4.67 x 10（-5）pg/细胞+/- 3.90 x 10（6）pg/细胞;对照：3.99 x 10（-6）pg/细胞+/- 5.50 x 10（7）pg/细胞）。当NHEK细胞暴露于300 μ M HD时，细胞悬液中存在的IL-8和TNF-α的量分别增加至对照水平的59倍和4倍。NHEK暴露于300 μ M HD对IL-1 β的释放具有高度可变的影响，其中有时IL-1 β的分泌增加到基线水平以上，而其他时间在细胞悬浮液中减少。暴露于100和300 μ M后24小时，从细胞培养瓶中收集上清液，观察到IL-6水平显著增加。IL-6以浓度依赖性方式释放，在上清液中分别为3.6倍至8.4倍。这些促炎介质IL-1 β、IL-8、TNF-α和IL-6可能在HD损伤中起重要作用。目前的研究结果表明，细胞因子的变化检测可作为潜在的生物标志物的皮肤水疱性损伤。
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-l beta (IL-1 beta), interleukin-6 (IL-6), interleukin-8 (IL-8), and tumor necrosis factor-alpha (TNF-alpha) 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 x 10(6) cells/mL) were exposed to HD (100 and 300 mu M) in keratinocyte growth medium (KGM) for 24 h at 37 degrees 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 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) pg/cell +/- 7.3 x 10 (7) pg/cell; control: 4.04 x 10 (6) +/- 2.80 x 10 (7) pg/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 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 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 suspensions of NHEK increased levels of IL-6 after exposure to 300 mu M HD (4.67 x 10 (-5) pg/cell +/- 3.90 x 10 (6) pg/cell; control: 3.99 x 10(-6) pg/cell +/- 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 NHEK 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 and other times decreased in cell suspensions. Supernatants were collected from cell culture flasks 24 h after exposure of 100 and 300 mu M 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-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 cutaneous vesicant injury.