Uncontrolled inflammation contributes to cutaneous damage following exposure to the warfare agent bis(2-chloroethyl) sulfide (sulfur mustard, SM). Activation of the p38 mitogen activated protein kinase (MAPK) precedes SM-induced cytokine secretion in normal human epidermal keratinocytes (NHEK). This study examined the role of p38-regulated MAPK activated kinase 2 (MK2) during this process. Time course analysis studies using NHEK cells exposed to 200 μM SM demonstrated rapid MK2 activation via phosphorylation that occurred within 15 minutes. p38 activation was necessary for MK2 phosphorylation as determined by studies using the p38 inhibitor SB203580. To compare the role of p38 and MK2 during SM-induced cytokine secretion, small interfering RNA (siRNA) targeting these proteins was utilized. TNF-α, IL-1β, IL-6 and IL-8 secretion was evaluated 24 hours postexposure, while mRNA changes were quantified after 8 hrs. TNF-α, IL-6 and IL-8 up regulation at the protein and mRNA level was observed following SM exposure. IL-1β secretion was also elevated despite unchanged mRNA levels. p38 knockdown reduced SM-induced secretion of all the cytokines examined, whereas significant reduction in SM-induced cytokine secretion was only observed with TNF-α and IL-6 following MK2 knockdown. Our observations demonstrate potential activation of other p38 targets in addition to MK2 during SM-induced cytokine secretion.
[Show abstract][Hide abstract] ABSTRACT: In clinical studies, the findings on sulfur mustard (SM) toxicity for CD3(+)CD4(+) and CD3(+)CD8(+) T lymphocyte subsets are contradictory. In animal experiments, the effect of SM on the T cell number and proliferation is incompatible and is even the opposite of the results in human studies. In this study, we observed the dynamic changes of T lymphocytes in the first week in a high-dose SM-induced model.
Mice were exposed to SM by subcutaneous injection (20 mg/kg) and were sacrificed 4 h, 24 h, 72 h and 168 h later. Spleen T lymphocyte proliferation was evaluated by (3)H-TdR. Flow cytometric analysis was used to observe the percentage of CD3(+)CD4(+) and CD3(+)CD8(+) T lymphocyte subsets. The IL-1β, IL-6, IL-10 and TNF-α levels in plasma were assayed using the Luminex method. DNA damage in bone marrow cells was observed with the single cell gel electrophoresis technique (SCGE).
SM continuously inhibited the proliferation of lymphocytes for 7 days, and there was a significant rebound of Con A-induced T lymphocyte proliferation only at 24 h. The percentage of CD3(+)CD4(+) and CD3(+)CD8(+) lymphocytes was upregulated, which was accompanied by increased IL-1β and TNF-α and decreased IL-10. The IL-6 level was gradually decreased in the PG group at 4 h. The peak of lymphocytic apoptosis and DNA damage occurred at 24 h and 72 h, respectively.
Our results show that SM significantly inhibited T lymphocyte proliferation as well as induced CD3(+)CD4(+) and CD3(+)CD8(+) upregulation. SM intoxication also significantly increased the levels of pro-inflammatory cytokines (IL-1β, IL-6 and TNF-α) and inhibited the level of anti-inflammatory cytokine IL-10. Our results may partly be due to the significant SM induced significant apoptosis and necrosis of lymphocytes as well as DNA damage of bone marrow cells. The results provided a favorable evaluation of SM immune toxicity in an animal model.
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