Wang Y, Zhao M-H, Zhang Y-K, Li X-M, Wang H-Y
Renal Division & Institute of Nephrology, Peking University First Hospital, Beijing, China.
Clin Exp Immunol. 2004 Apr;136(1):168-75. doi: 10.1111/j.1365-2249.2004.02408.x.
IgA deposition in glomerular mesangium and the interaction with mesangial cells may well be the final common pathway to IgA nephropathy (IgAN). Altered hinge-region O-glycosylation of IgA1 from patients with IgAN may predispose to mesangial deposition and activation of the mesangial cell (MC) by IgA1, via a novel IgA1 receptor, and may be a key event in the pathogensis of IgAN. The aim of this study was to investigate the binding capacity and biological effects of IgA1, from both patients with IgAN and healthy controls, on human mesangial cells (HMC). Serum IgA1 was isolated with jacalin affinity chromatography, heated to aggregated form (aIgA1) and labelled with (125)I. Binding capacity of aIgA1 in vitro to cultured primary HMC was evaluated by a radioligand binding assay and the specificity of binding was determined by a competitive inhibition assay. Intracellular calcium release was studied by confocal analysis and phosphorylation of extracellular signal-regulated kinase (ERK) was determined by Western blot analysis. Change of cell cycles was demonstrated by flow cytometry and HMC proliferation was evaluated by direct cell count. Expression of TGF-beta mRNA and production of supernatant fibronectin were tested by RT-PCR and indirect competitive ELISA, respectively. aIgA1 from both the patients with IgAN and normal controls bound to HMC in a dose-dependent, saturable manner, and was saturated at approximately 500 pmoles per 0.5 ml of aIgA1. aIgA1 from patients with IgAN, however, bound to HMC at a higher speed and Scatchard analysis revealed a Kd of (8.89 +/- 2.1) x 10(-8)m versus (4.3 +/- 1.2) x 10(-7)m for aIgA1 from healthy controls (P = 0.026). The binding was specific because it was only inhibited by unlabelled Mono-IgA1 (mIgA1) and not by serum albumin or IgG. aIgA1 from patients with IgAN could induce release of intracellular calcium, phosphorylation of ERK, DNA synthesis, proliferation of HMC, expression of TGF-betamRNA and secretion of fibronectin in HMC in a similar time-dependent manner as aIgA1 from healthy controls, but the effects were much stronger and the durations were much longer (P < 0.05, respectively). We conclude that aIgA1 from patients with IgAN has a higher binding capacity to HMC and stronger biological effects than aIgA1 from healthy controls. This suggests that direct interaction between IgA1 and HMC and subsequential pathophysiological responses may play an important role in the pathogenesis for IgAN.
IgA在肾小球系膜中的沉积以及与系膜细胞的相互作用很可能是IgA肾病（IgAN）的最终共同途径。IgAN患者IgA1铰链区O-糖基化改变可能通过一种新型IgA1受体，使IgA1易于在系膜沉积并激活系膜细胞（MC），这可能是IgAN发病机制中的关键事件。本研究的目的是探讨来自IgAN患者和健康对照者的IgA1对人系膜细胞（HMC）的结合能力和生物学效应。用红豆蔻凝集素亲和层析法分离血清IgA1，加热形成聚集形式（aIgA1）并用（125）I标记。通过放射配体结合试验评估体外培养的原代HMC中aIgA1的结合能力，并通过竞争性抑制试验确定结合的特异性。通过共聚焦分析研究细胞内钙释放，通过蛋白质印迹分析测定细胞外信号调节激酶（ERK）的磷酸化。通过流式细胞术证明细胞周期变化，通过直接细胞计数评估HMC增殖。分别通过RT-PCR和间接竞争ELISA检测TGF-βmRNA的表达和上清液纤连蛋白的产生。来自IgAN患者和正常对照者的aIgA1均以剂量依赖性、可饱和的方式与HMC结合，每0.5ml aIgA1约500皮摩尔时达到饱和。然而，来自IgAN患者的aIgA1与HMC的结合速度更快，Scatchard分析显示其解离常数（Kd）为（8.89±2.1）×10^(-8)m，而健康对照者的aIgA1为（4.3±1.2）×10^(-7)m（P = 0.026）。这种结合是特异性的，因为它仅被未标记的单IgA1（mIgA1）抑制，而不被血清白蛋白或IgG抑制。来自IgAN患者的aIgA1能够以与来自健康对照者的aIgA1类似的时间依赖性方式诱导HMC细胞内钙释放、ERK磷酸化、DNA合成、HMC增殖、TGF-βmRNA表达和纤连蛋白分泌，但效应更强且持续时间更长（P均<0.05）。我们得出结论，来自IgAN患者的aIgA1比来自健康对照者的aIgA1对HMC具有更高的结合能力和更强的生物学效应。这表明IgA1与HMC之间的直接相互作用以及随后的病理生理反应可能在IgAN的发病机制中起重要作用。
