Immunohistochemical distribution and subcellular localization of three distinct specific molecular structures of advanced glycation end products in human tissues

Using three mouse anti-human monoclonal antibodies for advanced glycation e nd products (AGEs), 6D12, 1F6, and 2A2, we examined the immunohistochemical distribution and localization of AGEs in various organs and tissues obtain ed from nondiabetic autopsy or biopsy cases (men and women, 41 to 86 years of age). 6D12 recognizes N-epsilon-(carboxymethyl)lysine (CML), a nonfluore scent and non-cross-linked AGE structure, and 1F6 recognizes fluorolink, a fluorescent and cross-linked AGE structure. The epitope of 2A2 is unknown b ut is different from that of CIVIL and fluorolink or other known AGE struct ures such as pyrraline, pentosidine, and crosslines. Immunohistochemistry w ith these monoclonal antibodies revealed the intra- and extracellular accum ulation of AGEs in these organs and tissues. By double immunohistochemical staining with two of the three monoclonal antibodies in different combinati ons, positive reaction products for all three monoclonal antibodies were de monstrated in macrophages widely distributed in various organs and tissues; endothelial cells of endocardium, arteries, veins, and blood capillaries; mesenchymal cells; epithelial or parenchymal cells; blood cells; and extrac ellular matrix. This result indicates that these three different AGE-specif ic molecules are formed intracellularly and extracellularly. In some cell t ypes, however, one or two of these specific molecules were not always found together, suggesting that the molecular structures of AGEs and their forma tion are heterogeneous. Immunoelectron microscopy demonstrated the localiza tion of AGE-labeled immunogold particles in the nuclei, nuclear envelope, m itochondria, endoplasmic reticula, Golgi complexes, endocytic vesicles, lys osomal vacuoles or granules, secretory granules, cytosol, and cell membrane s, as well as in the extracellular matrix. In addition, the double histoche mical staining method for ceroid/lipofuscin and immunohistochemistry for AG Es demonstrated intralysosomal formation and accumulation of AGEs in ceroid /lipofuscin pigments. These results suggest that the extracellularly produc ed AGEs are taken up by receptors into the cells and accumulate in secondar y lysosomes and that AGEs are formed intranuclearly and/or intracellularly, probably via different metabolic pathways.