Inflammatory but not apoptotic death of granulocytes citrullinates fibrinogen.
Inflammatory but not apoptotic death of granulocytes citrullinates fibrinogen.
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DOI：
10.1186/s13075-015-0890-0
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发表时间：
2015-12-17
影响因子：
4.9
通讯作者：
Orange DE
中科院分区：
文献类型：
--
作者：
Blachère NE;Parveen S;Fak J;Frank MO;Orange DE
神经元活化诱导抗瓜氨酸肽抗体（ACPA）的细胞内靶点的瓜氨酸化，其对类风湿性关节炎（RA）具有特异性。瓜氨酸化纤维蛋白原与ACPA结合，但对细胞外蛋白如何被瓜氨酸化的了解较少。产生纤维蛋白原的细胞肝细胞不表达肽基精氨酸脱亚胺酶（PAD），PAD酶也不包括N-末端信号肽以将其引导到分泌途径中。我们假设垂死的中性粒细胞在细胞外间隙释放PAD，这可能导致与RA相关的靶细胞外抗原（如纤维蛋白原）的瓜氨酸化。用全反式维甲酸（ATRA）诱导HL 60细胞分化为嗜酸性粒细胞样细胞。通过CD 11b染色、PAD 4、PAD 2和髓过氧化物酶表达、细胞分裂和细胞核形态确认分化。用各种刺激诱导死亡，包括冻融诱导坏死，离子霉素和PMA诱导NETosis，以及UV-B诱导凋亡。通过免疫组织化学和流式细胞术评估死亡标志物。为了定量细胞外瓜氨酸，将垂死的ATRA分化的HL 60细胞与纤维蛋白原一起培养24小时，并通过蛋白质印迹法探测上清液中的瓜氨酸纤维蛋白原、PAD 2和PAD 4。虽然NETotic和坏死的ATRA分化的HL 60细胞瓜氨酸化纤维蛋白原，凋亡细胞不瓜氨酸化纤维蛋白原，即使允许进行二次坏死。坏死的中性粒细胞裂解物与纤维蛋白原孵育也会导致纤维蛋白原瓜氨酸化。经ATRA分化的HL 60细胞经历坏死和NETotic死亡，但不发生凋亡或继发性坏死细胞死亡，通过蛋白质印迹法检测到PAD 2和PAD 4。我们牵连粒细胞经历炎性细胞死亡作为改变细胞外自身蛋白的机制，可能是与炎症性疾病，如类风湿性关节炎相关的自身免疫的目标。本文的在线版本（doi：10.1186/s13075-015-0890-0）包含补充材料，可供授权用户使用。
Neutrophil activation induces citrullination of intracellular targets of anticitrullinated peptide antibodies (ACPA), which are specific for rheumatoid arthritis (RA). Citrullinated fibrinogen is bound by ACPA but it is less well understood how extracellular proteins are citrullinated. The cells that produce fibrinogen, hepatocytes, do not express peptidyl arginine deiminase (PAD) enzymes nor do PAD enzymes include N-terminal signal peptides to direct them into the secretory pathway. We hypothesized that dying neutrophils release PAD in the extracellular space, and that this could cause citrullination of target extracellular antigens relevant to RA such as fibrinogen. HL60 cells were differentiated into neutrophil-like cells by treatment with all-trans retinoic acid (ATRA). Differentiation was confirmed by CD11b staining, PAD4, PAD2 and myeloperoxidase expression, cell division, and nuclear morphology. Death was induced with various stimuli, including freeze-thaw to induce necrosis, Ionomycin and PMA to induce NETosis, and UV-B to induce apoptosis. Death markers were assessed by immunohistochemistry and flow cytometry. To quantify extracellular citrullination, dying ATRA-differentiated HL60 cells were cultured with fibrinogen for 24 hours and supernatants were probed for fibrinogen citrullination, PAD2 and PAD4 by western blot. While both NETotic and necrotic ATRA differentiated HL60 cells citrullinated fibrinogen, apoptotic cells did not citrullinate fibrinogen, even when allowed to undergo secondary necrosis. Incubation of necrotic neutrophil lysates with fibrinogen also causes fibrinogen citrullination. PAD2 and PAD4 were detected by western blot of supernatants of ATRA-differentiated HL60 cells undergoing necrotic and NETotic death, but not apoptotic or secondarily necrotic cell death. We implicate granulocytes undergoing inflammatory cell death as a mechanism for altering extracellular self-proteins that may be targets of autoimmunity linked to inflammatory diseases such as rheumatoid arthritis. The online version of this article (doi:10.1186/s13075-015-0890-0) contains supplementary material, which is available to authorized users.