University of Zagreb, Faculty of Pharmacy and Biochemistry, Department of Medical Biochemistry and Hematology, Zagreb, Croatia.
Fidelta Ltd., Zagreb, Croatia.
Mol Immunol. 2019 Jul;111:53-63. doi: 10.1016/j.molimm.2019.04.002. Epub 2019 Apr 11.
Extracellular Hsp70 (eHsp70) can act as pro-inflammatory mediator and is elevated in blood of chronic obstructive pulmonary disease (COPD) patients. Most of those patients are smokers, and it was suggested previously that cigarette smoke might induce Hsp70 secretion from the circulating cells. Therefore, we aimed to explore inflammation-associated effects of cigarette smoke extract (CSE) and its combinations with eHsp70 in monocyte-derived macrophages (MDMs) and THP-1 cell line, used as systemic component models of COPD. We hypothesized that eHsp70 induces inflammation, but that it can also modulate cigarette smoke extract (CSE)-stimulated inflammatory responses. We assessed IL-8 secretion, TLR2, TLR4 and Hsp70 expressions, MAPKs and NF-κB activation, and cytotoxicity after treating the cells with CSE (2.5 and 5%) and its combinations with low-endotoxin recombinant human (rh) Hsp70, used to mimic eHsp70 effects. CSE induced IL-8 secretion from both cell types, but its combinations with rhHsp70 increased IL-8 release compared to CSE alone only from MDMs. In THP-1, combinations of rhHsp70 with 2.5% CSE induced TLR2 and TLR4 mRNA, while 5% CSE decreased TLR2 expression. In MDMs, CSE alone attenuated TLR2, while rhHsp70 increased TLR2 and lowered TLR4 gene expression. Hsp70 mRNA expression was suppressed in THP-1 with rhHsp70 and CSE; however, the same treatments increased its level in MDMs. CSE had cytotoxic effect only on MDMs, but cytotoxicity was reduced in co-treatments with rhHsp70, which also triggered apoptosis. CSE and rhHsp70 activated p38 and JNK, while ERK was activated only by rhHsp70 in MDMs. In THP-1, 2.5% CSE activated ERK, and 5% CSE activated p38. Inhibition of NF-κB and JNK in MDMs, and ERK and JNK in THP-1 cells, attenuated IL-8 release after rhHsp70 treatment. In conclusion, rhHsp70 provoked pro-inflammatory effects and could also modulate inflammatory response to CSE on protein and gene expression levels in THP-1 cells and MDMs, which suggests that eHsp70 might be implicated in systemic inflammation induced by cigarette smoke.
细胞外热休克蛋白 70（eHsp70）可作为促炎介质，在慢性阻塞性肺疾病（COPD）患者的血液中升高。大多数 COPD 患者是吸烟者，先前有研究表明，香烟烟雾可能会诱导循环细胞分泌 Hsp70。因此，我们旨在探索香烟烟雾提取物（CSE）及其与单核细胞衍生的巨噬细胞（MDM）和 THP-1 细胞系中 eHsp70 的组合对 COPD 的全身性成分模型的炎症相关影响。我们假设 eHsp70 诱导炎症，但它也可以调节香烟烟雾提取物（CSE）刺激的炎症反应。我们评估了细胞用 CSE（2.5%和 5%）及其与低内毒素重组人（rh）Hsp70 的组合处理后 IL-8 分泌、TLR2、TLR4 和 Hsp70 表达、MAPKs 和 NF-κB 激活以及细胞毒性，rhHsp70 用于模拟 eHsp70 的作用。CSE 诱导两种细胞类型的 IL-8 分泌，但与 rhHsp70 的组合仅从 MDM 中增加了与 CSE 相比的 IL-8 释放。在 THP-1 中，rhHsp70 与 2.5% CSE 的组合诱导 TLR2 和 TLR4 mRNA，而 5% CSE 降低 TLR2 表达。在 MDM 中，CSE 单独减弱 TLR2，而 rhHsp70 增加 TLR2 并降低 TLR4 基因表达。rhHsp70 和 CSE 处理下调了 THP-1 中的 Hsp70 mRNA 表达，但在 MDM 中增加了其水平。CSE 仅对 MDM 具有细胞毒性作用，但 rhHsp70 共同处理可降低细胞毒性，并触发细胞凋亡。CSE 和 rhHsp70 激活了 p38 和 JNK，而 ERK 仅在 MDM 中被 rhHsp70 激活。在 THP-1 中，2.5% CSE 激活了 ERK，而 5% CSE 激活了 p38。在 MDM 中抑制 NF-κB 和 JNK，以及在 THP-1 细胞中抑制 ERK 和 JNK，减轻了 rhHsp70 处理后的 IL-8 释放。总之，rhHsp70 引发了促炎作用，并且还可以调节 CSE 在 THP-1 细胞和 MDM 中对蛋白质和基因表达水平的炎症反应，这表明 eHsp70 可能参与了香烟烟雾引起的全身炎症。
