Adaptation Physiology Group, Department of Animal Sciences, Wageningen University, P.O. Box 338, 6700 AH Wageningen, The Netherlands.
Dev Comp Immunol. 2010 Aug;34(8):821-7. doi: 10.1016/j.dci.2010.03.002. Epub 2010 Mar 23.
Natural antibodies (NAb) perform many important functions in various immune responses and are often polyreactive of nature with low binding affinity. Natural auto-antibodies (N(A)Ab) are NAb binding at least one auto-antigen. Polyreactivity of N(A)Ab has been proposed to rest on post-translational polymorphism of the immunoglobulin F(ab)(2) fragment caused by various locally present oxidizing agents, salts and lower or higher pH. Challenge with pathogen-associated molecular patterns (PAMP), such as lipopolysaccharide (LPS) or lipoteichoic acid (LTA), respectively, may underlie N(A)Ab polymorphism by the activation of inflammatory cells whose products affect the three-dimensional structure of N(A)Ab F(ab)(2) fragments. We evaluated by Western blotting the effects of subcutaneous administered LPS and LTA, respectively, on binding characteristics of chicken N(A)Ab towards the 'auto-antigen' chicken-liver-cell-lysate (CCL) in situ prior to (day 0) and 3 days after subcutaneous challenge, as well as the effect of different in vitro maltreatments in the form of oxidizing agents: 5mM hydrogen peroxide, 10mM hydrogen peroxide, pH 2.6, and pH 2.0, aqua dest, and phosphate buffered saline (PBS) as control, respectively, on chicken N(A)Ab polymorphism. On both days 0 and 3 after challenge, N(A)Ab in plasma from all chickens bound to CCL. No significant differences of in vivo or in vitro maltreatments were found on the number of CCL fragments bound by the N(A)Ab. However, significant differences in the staining patterns of individual CCL molecular weight-identified fragments (MWIF) were found. The sum (Sigma) of newly stained fragments and disappeared fragments (SigmaMWIF) that were bound by plasma samples was significantly different between in vivo LPS or in vivo LTA challenged birds. Also, significant differences in the percentages of extinction intensity of these SigmaMWIF were found. In addition, the plasma samples obtained at day 0 and day 3 from both LTA and LPS challenged birds were all similarly prone to in vitro maltreatment. In vitro maltreatment with pH 2.0 had the highest effect on chicken N(A)Ab polymorphism, whereas aqua dest had the lowest effect. The change of CCL fragments recognized by chicken N(A)Ab was not caused by unmasking immune complexes. The present findings suggest that (1) N(A)Ab are present in chicken plasma, (2) chicken N(A)Ab are prone to irreversible post-translational polymorphism in vitro, and (3) post-translational polymorphism of chicken N(A)Ab can be initiated by PAMP-induced inflammatory agents in situ. The consequences of these finding are discussed.
天然抗体 (NAb) 在各种免疫反应中发挥着许多重要功能，通常具有低亲和力的多反应性。天然自身抗体 (N(A)Ab) 是指至少与一种自身抗原结合的 NAb。N(A)Ab 的多反应性据推测是由于各种局部存在的氧化剂、盐和较低或较高 pH 值引起的免疫球蛋白 F(ab)(2)片段的翻译后多态性所致。与病原体相关的分子模式 (PAMP)（如脂多糖 (LPS) 或脂磷壁酸 (LTA)）的接触分别可能通过激活炎性细胞来导致 N(A)Ab 多态性，炎性细胞的产物会影响 N(A)Ab F(ab)(2)片段的三维结构。我们通过 Western 印迹评估了皮下给予 LPS 和 LTA 分别对鸡 N(A)Ab 在皮下注射前（第 0 天）和第 3 天与原位“自身抗原”鸡肝细胞裂解物（CCL）结合特性的影响，以及不同体外应激处理形式的影响，包括 5mM 过氧化氢、10mM 过氧化氢、pH 2.6 和 pH 2.0、去离子水和磷酸盐缓冲盐水 (PBS)，分别作为对照，对鸡 N(A)Ab 多态性的影响。在挑战后的第 0 天和第 3 天，所有鸡的血浆 N(A)Ab 均与 CCL 结合。在体内或体外应激处理后，N(A)Ab 与 CCL 结合的 CCL 片段数量没有显著差异。然而，发现了个体 CCL 分子量鉴定片段 (MWIF) 的染色模式存在显著差异。与血浆样本结合的新染色片段和消失片段的总和 (SigmaMWIF) 在体内 LPS 或体内 LTA 处理的鸟类之间存在显著差异。此外，这些 SigmaMWIF 的消光强度百分比也存在显著差异。此外，来自 LTA 和 LPS 处理鸟类的第 0 天和第 3 天的血浆样本在体外应激处理下均具有相似的易感性。pH 2.0 的体外应激处理对鸡 N(A)Ab 多态性的影响最大，而去离子水的影响最小。鸡 N(A)Ab 识别的 CCL 片段的变化不是由于免疫复合物的脱掩。本研究结果表明，（1）N(A)Ab 存在于鸡血浆中，（2）鸡 N(A)Ab 易于体外发生不可逆的翻译后多态性，（3）PAMP 诱导的原位炎性物质可启动鸡 N(A)Ab 的翻译后多态性。讨论了这些发现的后果。
