Renal Division, Department of Medicine, Peking University, First Hospital, Peking University Institute of Nephrology, Key Laboratory of Renal Disease, Ministry of Health of China, Key Laboratory of Chronic Kidney Disease Prevention and Treatment Peking University, Ministry of Education, Peking-Tsinghua Center for Life Sciences, Beijing 100034, China.
Mol Immunol. 2013 May;54(1):68-73. doi: 10.1016/j.molimm.2012.10.041. Epub 2012 Nov 28.
C5a and the neutrophil C5a receptor play a central role in antineutrophil cytoplasmic antibody (ANCA)-mediated neutrophil recruitment and activation. Our recent study found that activation of p38 mitogen-activated protein kinase (p38MAPK), extracellular signal-regulated kinase (ERK) and phosphoinositol 3-kinase (PI3K) are all important steps in the translocation of ANCA antigens by C5a-mediated priming and activation of neutrophils. The current study further investigated the protein kinase C (PKC) pathway of C5a-mediated neutrophils for ANCA-induced activation. The effect of the PKC inhibitor (bisindolylmaleimide I, BIS) was tested on respiratory burst and degranulation of C5a-primed neutrophils activated with ANCA, as well as on C5a-induced increase in expression of PR3 and MPO. For C5a-primed neutrophils for MPO-ANCA-induced respiratory burst, the mean fluorescence intensity (MFI) value was 369.8±18.8, which decreased to 308.3±14.2 upon pre-incubation with BIS (P<0.001). For PR3-ANCA-positive IgG, the MFI value increased in C5a-primed neutrophils, which decreased upon pre-incubation with BIS. The lactoferrin concentration increased from 414.8±26.9 ng/ml in the non-primed neutrophils supernatant to 1099.8±80.7 ng/ml in C5a-primed neutrophils induced by MPO-ANCA-positive IgG supernatant (P<0.001), and decreased to 814.5±45.3 ng/ml upon pre-incubation with BIS (P<0.01). The lactoferrin concentration also increased in C5a-primed neutrophils induced by PR3-ANCA-positive IgG supernatant and decreased upon pre-incubation with BIS. Membrane expression of PR3 (mPR3) expression increased from 788.0±87.5 in untreated cells to 1071.3±81.3 after C5a treatment and decreased to 827.3±48.1 by BIS (P<0.05). Activation of PKC is an important step in the translocation of ANCA antigens and C5a-induced activation of neutrophils by ANCA.
C5a 和中性粒细胞 C5a 受体在抗中性粒细胞胞浆抗体 (ANCA) 介导的中性粒细胞募集和激活中发挥核心作用。我们最近的研究发现，p38 丝裂原活化蛋白激酶 (p38MAPK)、细胞外信号调节激酶 (ERK) 和磷酸肌醇 3-激酶 (PI3K) 的激活都是 C5a 介导的 ANCA 抗原转位的重要步骤，通过 C5a 介导的中性粒细胞的初始激活。本研究进一步研究了 C5a 介导的中性粒细胞的蛋白激酶 C (PKC) 途径，用于 ANCA 诱导的激活。测试了 PKC 抑制剂 (双吲哚马来酰亚胺 I，BIS) 对 C5a 预激活的中性粒细胞与 ANCA 激活后呼吸爆发和脱颗粒的影响，以及 C5a 诱导的 PR3 和 MPO 表达增加的影响。对于 C5a 预激活的中性粒细胞，MPO-ANCA 诱导的呼吸爆发，平均荧光强度 (MFI) 值为 369.8±18.8，用 BIS 预处理后降至 308.3±14.2(P<0.001)。对于 PR3-ANCA 阳性 IgG，C5a 预激活的中性粒细胞中 MFI 值增加，用 BIS 预处理后降低。乳铁蛋白浓度从非预激活的中性粒细胞上清液中的 414.8±26.9ng/ml 增加到 MPO-ANCA 阳性 IgG 上清液诱导的 C5a 预激活的中性粒细胞中的 1099.8±80.7ng/ml(P<0.001)，用 BIS 预处理后降至 814.5±45.3ng/ml(P<0.01)。PR3-ANCA 阳性 IgG 上清液诱导的 C5a 预激活的中性粒细胞中乳铁蛋白浓度也增加，用 BIS 预处理后降低。PR3 的膜表达 (mPR3) 表达从未经处理的细胞中的 788.0±87.5 增加到 C5a 处理后的 1071.3±81.3，并通过 BIS 降至 827.3±48.1(P<0.05)。PKC 的激活是 ANCA 抗原转位和 C5a 诱导的 ANCA 激活中性粒细胞的重要步骤。
