受損細胞所釋放之內源性危險因子引發嗜中性白血球胞外網狀結構

Abstract

嗜中性白血球胞外網狀結構 (neutrophil extracellular traps, NETs) 是當嗜中性白血球和細菌或其他刺激物接觸後，會釋放出由 DNA、histones、granular proteins 等物質所構成的網狀結構，使嗜中性白血球得以抓住病原菌進而將之清除。而嗜中性白血球產生 NETs 之後，最終會使其走向死亡，此過程又稱作 ”NETosis”。除了抗菌的作用，有許多研究也指出 NETs 的產生和組織損傷以及自體免疫疾病相關。而在一般情況下，不論人體有無受到感染，一旦組織發生損傷受損細胞就會釋放出內源性損傷相關分子 (damage-associated molecular patterns, DAMPs)，這些 DAMPs 會吸引嗜中性白血球到達受傷部位，引起急性發炎反應。本研究的目的，是為了探討在無菌發炎的情形下，這些由受損細胞所釋放出的內源性危險因子是否會刺激嗜中性白血球胞外網狀結構的產生。我們的實驗結果顯示，人類嗜中性白血球經過不同壞死細胞 (HL60, A549, Hep3B) 的刺激後，會因此而活化產生活性氧分子 (reactive oxygen species, ROS)，且 p38 MAPK 和 ERK 的磷酸化程度也會上升，這代表了嗜中性白血球的活化。在刺激過後一段時間，除了於螢光顯微鏡下可觀察到，經壞死細胞處理後的嗜中性白血球會釋放出由網狀 DNA 和 elastase 構成的 NETs，也藉由偵測 citrullinated histone H3 確認了 NETs 的形成。此外，壞死細胞所誘導產生的 NETs 會因為 IL-8、GM-CSF、TNF-α 等發炎相關細胞激素的共處理而使此現象增強。另一方面，我們的結果顯示，NETs 的產生會因為 ROS、ERK 和 p38 MAPK 的抑制劑 (分別為 diphenyleneiodonium (DPI), U0126, SB203580) 存在而減緩。 Histones 是已知由受損細胞所釋放出的DAMPs。我們發現，純化後的 histone mixture (含有 histones H1, H2, H3 和 H4) 能夠刺激嗜中性白血球透過 MAPK 磷酸化以及誘導 ROS 的產生，最終使 NETs 形成。而經 histones 誘導所產生的 NETs 會因 DPI、U0126、SB203580 和 TLR4 拮抗劑 TAK-242 的處理而受到抑制，表示 histones 是透過 TLR4 來刺激嗜中性白血球。本研究顯示在無菌發炎的情形下，嗜中性白血球會受到受損細胞所釋放之內源性危險因子刺激，並產生嗜中性白血球胞外網狀結構，而 histones 和 DAMPs 是透過 TLR4 刺激嗜中性白血球。為了解嗜中性白血球是受到哪一種 histone(s) (H1, H2, H3, 或是 H4) 才誘發 NETs 的產生，及其在無菌發炎情形下所扮演之腳色對於體內平衡的維持及疾病產生的影響，需要更進一步的探討和實驗。

Neutrophil extracellular traps (NETs), which form when neutrophils are in contact with microbes, are extracellular structures composed of DNA, histones, and granular and cytoplasmic proteins that trap and kill pathogens. Neutrophils forming NETs also undergo a form of cell death termed “NETosis.” In addition to the antimicrobial effect, NETs are also associated with tissue damage and certain autoimmune diseases. It is known that upon tissue damage, even in the absence of infection, the injured cells can release endogenous damage-associated molecular patterns (DAMPs) that trigger an acute inflammatory response characterized by an influx of neutrophils to the injured site. The aim of this study is to investigate whether DAMPs can stimulate NET formation during sterile inflammation. Stimulation of purified human blood neutrophils with freeze-thawed necrotic HL60, A549, or Hep3B cells resulted in the the phosphorylation ERK and p38 MAPK and production of reactive oxygen species (ROS), which are indicatives of neutrophil activation. At a later time after stimulation, neutrophils with web-like DNA structures and extracellular elastase staining were observed under the immunofluorescence microscope, indicating the formation of NETs. NET formation was also confirmed by the detection of citrullinated histone H3 in neutrophils. Necrotic cell-induced NET formation could be further enhanced when neutrophils were co-treated with proinflammatory cytokines IL-8, GM-CSF, or TNF-α. On the other hand, NET formation was attenuated in the presence of inhibitors of ERK (U0126), p38 MAPK (SB203580), or ROS (diphenyleneiodonium, DPI). Histones are known DAMPs that are released from injured cells. We found that a purified histone mixture (containing histones H1, H2, H3 and H4) could stimulate MAPK (p38 and ERK) phosphorylation, ROS production, and NET formation in neutrophils. The induction of NETs by histones could be blocked by U0126, SB203580, DPI, and the TLR4 antagonist TAK-242, indicating that histones stimulate neutrophils via TLR4. Taken together, our results demonstrate that during sterile inflammation, neutrophils can be activated by endogenous danger signals to form NETs, and histones are DAMPs that can directly stimulate NET formation via TLR4 activation. Further investigation will be carried out to identify which histone(s) (H1, H2, H3, or H4) exhibit the NET-inducing activity, and to elucidate how NET formation may affect tissue homeostasis and/or pathogenesis in sterile inflammation.