Wang Ying-Ying, Schroeder Holly A, Nunn Kenetta L, Woods Karen, Anderson Deborah J, Lai Samuel K, Cone Richard A
Department of Biophysics, Johns Hopkins University, Baltimore, Maryland, United States of America.
Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina - Chapel Hill, Chapel Hill, North Carolina, United States of America.
PLoS One. 2016 Jun 30;11(6):e0158338. doi: 10.1371/journal.pone.0158338. eCollection 2016.
Human cervicovaginal mucus (CVM) is a viscoelastic gel containing a complex mixture of mucins, shed epithelial cells, microbes and macromolecules, such as antibodies, that together serve as the first line of defense against invading pathogens. Here, to investigate the affinity between IgG and different mucus constituents, we used Fluorescence Recovery After Photobleaching (FRAP) to measure the diffusion of IgG in fresh, minimally modified CVM. We found that CVM exhibits substantial spatial variations that necessitate careful selection of the regions in which to perform FRAP. In portions of CVM devoid of cells, FRAP measurements using different IgG antibodies and labeling methods consistently demonstrate that both exogenous and endogenous IgG undergo rapid diffusion, almost as fast as in saline, in good agreement with the rapid diffusion of IgG in mid-cycle endocervical mucus that is largely devoid of cells. This rapid diffusion indicates the interactions between secreted mucins and IgG must be very weak and transient. IgG also accumulated in cellular debris and shed epithelial cells that had become permeable to IgG, which may allow shed epithelial cells to serve as reservoirs of secreted IgG. Interestingly, in contrast to cell-free regions of CVM, the diffusion of cell-associated IgG was markedly slowed, suggesting greater affinity between IgG and cellular constituents. Our findings contribute to an improved understanding of the role of IgG in mucosal protection against infectious diseases, and may also provide a framework for using FRAP to study molecular interactions in mucus and other complex biological environments.
人宫颈阴道黏液（CVM）是一种粘弹性凝胶，含有黏蛋白、脱落的上皮细胞、微生物和大分子（如抗体）的复杂混合物，它们共同构成抵御入侵病原体的第一道防线。在此，为了研究IgG与不同黏液成分之间的亲和力，我们使用光漂白后荧光恢复（FRAP）技术来测量IgG在新鲜的、经过最小程度修饰的CVM中的扩散。我们发现CVM表现出显著的空间差异，这就需要仔细选择进行FRAP测量的区域。在CVM中无细胞的部分，使用不同IgG抗体和标记方法进行的FRAP测量一致表明，外源性和内源性IgG都能快速扩散，几乎与在盐溶液中的扩散速度一样快，这与IgG在细胞含量很少的月经周期中期宫颈黏液中的快速扩散情况相符。这种快速扩散表明分泌的黏蛋白与IgG之间的相互作用一定非常微弱且短暂。IgG还会在细胞碎片和对IgG具有通透性的脱落上皮细胞中积累，这可能使脱落上皮细胞成为分泌型IgG的储存库。有趣的是，与CVM的无细胞区域相比，与细胞相关的IgG的扩散明显减慢，这表明IgG与细胞成分之间的亲和力更强。我们的研究结果有助于更好地理解IgG在黏膜抗感染疾病保护中的作用，也可能为利用FRAP研究黏液和其他复杂生物环境中的分子相互作用提供一个框架。
