Abstract：
Bullous pemphigoid (BP), an autoimmune subepidermal blistering disease, shows tense blisters associated with urticarial erythema. Tissue-bound Immunoglobulin G (IgG) at the basement membrane zone (BMZ) detected by direct immunofluorescence (DIF) is strong evidence for a diagnosis of BP. The sensitivity of DIF is higher in complement component 3 (C3) than in IgG, but the reason for this different sensitivity is not fully understood. In this study, we performed several ex vivo studies to investigate the possible mechanism of IgG negativity and C3 positivity at the BMZ by DIF in some BP cases. First, sera from BP patients showing IgG negativity by DIF were found to clearly react to the BMZ in their own DIF skin samples. Next, indirect immunofluorescence (IIF) was performed using sera diluted with different pH phosphate-buffered saline (PBS), pH 7.4, 6.0, and 3.0. Patients\' sera diluted with pH 7.4 PBS showed linear staining at the BMZ, but sera diluted with pH 6.0 PBS and pH 3.0 PBS showed lower fluorescence intensities. Finally, sections of skin from BP patients were pre-incubated with different pH PBS (pH 3.0, 6.0, and 7.4), followed by staining with anti-human IgG and C3. The fluorescence intensities were notably lower for IgG and C3 that had been pre-incubated with pH 3.0 PBS and pH 6.0 PBS than for IgG and C3 that had been pre-incubated with pH 7.4 PBS. These results suggest that a low pH condition hinders the binding of autoantibodies to the BMZ, that is, the drop in tissue pH induced by inflammation inhibits autoantibodies from depositing at the BMZ. Furthermore, the drop in tissue pH causes tissue-bound autoantibodies to detach from the BMZ. Complement fragments are activated not only on IgG but also on the cell surface of cells close to IgG during complement activation. IgG may detach from the BMZ under low pH condition induced by inflammation, but some complement fragments remain at the BMZ. These phenomena may help to explain why C3 is more sensitive than IgG when DIF is used to diagnose BP.
摘要：
大疱性类天疱疮（BP），一种自身免疫性表皮下起泡疾病，显示与荨麻疹红斑相关的紧张水泡。通过直接免疫荧光（DIF）检测到的基底膜区（BMZ）处的组织结合免疫球蛋白G（IgG）是诊断BP的有力证据。补体成分3（C3）的DIF敏感性高于IgG，但是这种不同敏感性的原因还没有完全理解。在这项研究中,我们进行了几项离体研究,以研究在某些BP病例中,通过DIF在BMZ出现IgG阴性和C3阳性的可能机制.首先,发现通过DIF显示IgG阴性的BP患者的血清在他们自己的DIF皮肤样本中明显与BMZ反应。接下来,使用用不同pH的磷酸盐缓冲盐水（PBS）稀释的血清进行间接免疫荧光（IIF），pH7.4、6.0和3.0。用pH7.4PBS稀释的患者血清在BMZ显示线性染色，但用pH6.0PBS和pH3.0PBS稀释的血清显示较低的荧光强度。最后,将BP患者的皮肤切片用不同pH的PBS（pH3.0、6.0和7.4）预孵育，然后用抗人IgG和C3染色。已经用pH3.0PBS和pH6.0PBS预温育的IgG和C3的荧光强度显著低于已经用pH7.4PBS预温育的IgG和C3的荧光强度。这些结果表明，低pH条件阻碍了自身抗体与BMZ的结合，也就是说,炎症诱导的组织pH下降抑制了自身抗体在BMZ的沉积。此外，组织pH值的下降导致组织结合的自身抗体与BMZ分离。补体片段在补体激活期间不仅在IgG上被激活,而且在接近IgG的细胞的细胞表面上被激活。在炎症引起的低pH条件下，IgG可能会从BMZ中脱离。但BMZ还保留了一些补体片段.这些现象可能有助于解释为什么当DIF用于诊断BP时C3比IgG更敏感。
