Immunofluorescence Deconvolution Microscopy and Image Reconstruction of Human Defensins in Normal and Burned Skin

Objective: The aim of this study was visualization and localization of the human antimicrobials human beta defensins 1, 2, and 3, neutrophil defensin alpha (human neutrophil peptide), and the cathelicidin LL-37 in normal and burned skin, and determination of the cell types in which these antimicrobials were localized. Methods: Tissue sections were probed with antimicrobial antibodies, tagged with fluorescently labeled secondary antibodies, and subjected to fluorescence deconvolution microscopy and image reconstruction. Images were generated by stacking multiple-section scans, which were then volume rendered by rotating stacks 360° about an axis, or modeled in 3 dimensions. Results: This technique yields a definitive image, providing a rapid basis for further quantification and manipulation from a full 3-dimensional aspect. In normal skin, human beta defensin-1 was localized to the perinuclear region of keratinocytes; human beta defensin-2 was primarily localized to the stratum germinativum; human beta defensin-3 was found in dendritic cells of the stratum spinosum; human neutrophil peptide was randomly distributed in the papillary dermis; and LL-37 was concentrated in the stratum corneum and along ducts. In burned skin, in which keratinocytes are lost or destroyed, human beta defensin-1 was present in dermal glandular structures including hair shafts; human beta defensin-2 and human beta defensin-3 were found in the remaining keratin layers and glands of the lower dermis; human neutrophil peptide was primarily localized to hair shafts, though visible in residual keratin layers; and LL-37 was evident in very high concentrations in the epithelium of sweat ducts. Conclusion: We conclude via this technique that cells in the lower dermal and subdermal regions of burned skin synthesize antimicrobials after burn injury, and maintain something of a barrier against infection. This methodology is discussed and explained in this article.