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 degrees 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.