SAA通过TLR-2/TLR-4/NF-κB通路诱导角质形成细胞IL-1β的表达

Serum Amyloid A Induces Interleukin-1β Secretion from Keratinocytes Via TLR-2,TLR-4 and NF-κB Pathways

目的探讨血清淀粉样蛋白A(SAA)在体外角质形成细胞白细胞介素(IL)-1β分泌中可能的作用。方法分离培养包皮的表皮角质形成细胞,分为空白对照组和SAA不同浓度刺激组(1μg/m L、10μg/m L、100μg/m L)。分别用Real-time PCR和ELISA检测细胞中IL-1β前体mRNA和成熟IL-1β的表达。角质形成细胞在SAA(10μg/m L)存在下与同种型对照免疫球蛋白Ig G1、TLR-2抗体(10μg/m L)、TLR-4抗体(20μg/m L)或TLR-2抗体+TLR-4抗体孵育24h。RT-PCR检测IL-1βmRNA的表达。将角质形成细胞暴露SAA 15min后,通过流式细胞术测定NF-κB p65磷酸化。在存在NF-κB抑制剂Bay11-7082的情况下,以10μg/m L SAA处理正常原代角质形成细胞。24h后,RT-PCR法检测细胞IL-1βmRNA水平。结果 SAA刺激角质形成细胞中IL-1β的表达。随SAA浓度的升高IL-1β的表达也逐渐增加。角质形成细胞在SAA(10μg/m L)存在下与同种型对照免疫球蛋白Ig G1、TLR-2抗体(10μg/m L),TLR-4抗体(20μg/m L)或TLR-2抗体+TLR-4抗体孵育24h,IL-1βmRNA表达被不同程度抑制。将正常原代角质形成细胞暴露于SAA后,NF-κB p65磷酸化表达增加。在存在NF-κB抑制剂Bay11-7082的情况下,SAA诱导IL-1βmRNA的表达明显被抑制。结论 SAA在体外角质形成细胞中通过TLR-2/TLR-4/NF-κB通路诱导IL-1β表达。

Objective To evaluate the potential roles of serum amyloid A SAA in the secretion of interleukin IL -1β in keratinocytes in vitro Methods The epidermal keratinocytes of the foreskin were isolated and cultured in Epilife with different concentration of SAA 1μg/mL,10μg/mL and 100μg/mL,respectively As a result,the three groups above and the control group constituted the research groups The levels of precursor mRNA pre-mRNA of IL-1β and mature IL-1β were determined by real-time PCR RT-PCR and ELISA respectively The neonatal foreskin-isolated normal primary keratinocytes were incubated in the presence of SAA 10μg/mL in combination with isotype control immunoglobulin Ig G1,TLR-2 antibody 10μg/mL ,TLR-4 antibody 20μg/mL or TLR-2 antibody TLR-4 antibody for 24hThe cells were then analysed for IL-1β mRNA levels by real-time transcription-polymerase chain reaction RT-PCR Normal primary keratinocytes were exposed to the indicated concentrations of SAA for 15minNuclear factor kappa B NF-κB p65 phosphorylation was determined by flow cytometry Normal primary keratinocytes were treated with SAA 10μg/mL ,in the presence NF-κB inhibitor Bay11-7082 at the indicated concentrations After 24h,the cells were analysed for IL-1β mRNA levels by RT-PCRResults SAA induces the transcription and secretion of interleukin IL -1β in human keratinocytes The production of mature IL-1β following stimulation with a range of concentrations of SAA was increased significantlyIL-1β mRNA was inhibited to different degrees when normal primary keratinocytes were incubated in the presence of SAA 10μg/mL in combination with isotype control IgG1,TLR-2 antibody 10μg/mL ,TLR-4 antibody 20μg/mL or TLR-2 antibody TLR-4 antibody for 24hPhosphorylated forms of NF-κB p65 were increased in SAA-stimulated keratinocytes Using Bay11-7082,an NF-κB inhibitor,we demonstrated that SAA-induced IL-1β mRNA was suppressed significantlyConclusionTLR-2/TLR-4/NF-κB signaling is involved in the SAA-mediated induction of IL-1β production in keratinocytes