Immunolocalization and Correlation Frequencies of Lingual Antimicrobial Peptide and Lactoferrin in Bovine Alveolar Epithelium and Bovine Mammary Gland

Mastitis is most critical disease in dairy cows and causes huge cost in the dairy industry. To prevent and treat it, it is important to understand the mechanisms of immune function in the mammary gland. Innate immunity is non-specific acute-response immune function. Some components of innate immunity in the mammary gland are found, e.g. Lingual Antimicrobial Peptide (LAP), Lactoferrin (LF). These components are found to be localized in the alveolar epithelium of mammary gland. LAP belongs to the β-defensin family, and plays a crucial role in killing a large variety of microorganisms. LF belongs to an iron-binding glycoprotein and has antibacterial activity. It is reported that LF has been localized immunohistochemically in mammary epithelial cells of lactating cows. Our previous study revealed that secretion of LAP into milk proceeded to that of LF after lipopolysaccharide (LPS) injection into the mammary gland. From this result, it is hypothesized that immunohistochemistry probably shows positive to either LF or LAP but not both in the alveolus vs epithelium in the mammary gland. Therefore, the aim of the present study is to investigate the immunolocalization of LAP and LF in the same bovine mammary tissue. Bovine mammary tissues were collected in the slaughterhouse and were fixed with neutralized formalin immediately. Paraffin sections (2-um thickness) were processed with antigen retrieval treatment followed by blocking with casein milk. Sections were cultured with LF antibody or LAP antibody. Immunoreaction products were visualized by incubation with a DAB. LAP and LF were localized in the cytoplasm of epithelial cell of alveolus. In some cases, LAP and LF were seen clearly in the same alveoli of section. In other cases, some epithelial cells were stained only LAP, but not LF, and other epithelial cells of alveolus were stained only LF, but not LAP. These results suggest the possibility that LAP and LF are differentially synthesized in the alveolar epithelium and may support our previous findings that their secretion occurs at the different time course.