Anat Cell Biol.  2013 Dec;46(4):246-253. 10.5115/acb.2013.46.4.246.

Ultrastructural immunolocalization of beta-defensin-27 in granulocytes of the dermis and wound epidermis of lizard suggests they contribute to the anti-microbial skin barrier

• Alibardi L

• KMID: 2046765
• DOI: http://doi.org/10.5115/acb.2013.46.4.246

Abstract

The high resistance to infections in lizard wounds suggests that these reptiles possess effective antimicrobial peptides in their tissues. The present immunocytochemical study shows the cellular localization of beta-defensin 27 in tail tissues and in the blood, a defensin previously identified in the lizard Anolis carolinensis through biomolecular methods. Beta-defensin-27 immunoreactivity is only observed in some large granules mainly contained in heterophilic granulocytes that are sparse within the dermis of the skin or in the isolated blood. This peptide is absent in other cell types of the skin, in keratinocytes and in subdermal muscle tissue of the tail in normal conditions. Pre-corneous keratinocytes of the regenerating tail epidermis are unlabeled or show a weak labeling for the peptide only in sparse cytoplasmic areas or in the extracellular spaces among corneocytes of the wound and regenerating epidermis. The study suggests that beta-defensin 27 is normally stored in granulocytes present in the blood or in connective tissues while in the epidermis keratinocytes do not show the presence of this peptide unless these cells are stimulated from injury to produce and likely release beta-defensins.

Keyword

Lizards; Skin; Blood; Beta-defensin-27; Immunolocalization

MeSH Terms

beta-Defensins
Connective Tissue
Cytoplasm
Dermis*
Epidermis*
Extracellular Space
Granulocytes*
Keratinocytes
Lizards*
Methods
Muscles
Peptides
Reptiles
Skin*
Tail
Wounds and Injuries*
Peptides
beta-Defensins

Figure

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  Fig. 1 Amino acid sequences of beta-defensins from the lizard Anolis carolinensis (AcBD15 and -27 [6]), the soft-shelled turtle Apalone spinifera (TuBD-1 [13]), the turtles Caretta caretta [10], Pelodiscus sinensis [11] and Emys orbicularis [12]. Note the characteristics positions of the 6 cysteines (▒) and the glycines (▓) localized two positions before the second (arrowhead) and the fourth (double arrowhead) cysteine.

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  Fig. 2 Histology of the apical region of a regenerating tail (A, B) and Ac-BD-27 immunoreactivity in sparse cells (C-F). (A) The blastema (bl) consists in a mesenchymal tissue surrounded by a thick wound epidermis (w). The arrows indicate the areas where granulocytes were seen using the electron microscope. (B) Detail of the wound epidermis (w) and the underlying blastema mesenchyme (bl). The arrows indicate the areas were granulocytes were later identified under the electron microscope. (C) Positive granular cells (arrow) in the inter-muscle connective of the tail stump. (D) Positive cells (arrow) located in the mesenchyme of the regenerating tail. (E) Immunofluorescent cells (arrows) in the wound epithelium (w) with its stratum corneum (c). Dashes indicate the dermal-epidermal boundary. (F) Detail of immunofluorescent granular cells localized in the dermis (arrowhead) and at the base (arrow) of the wound epidermis (w). Dashes indicate the dermal-epidermal boundary. Scale bars=100 µm (A), 20 µm (B), 10 µm (C-F).

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  Fig. 3 Ultrastructural view of a heterophilic granulocyte (A) and Ac-BD-27 immunoreactive granules from tissue granulocytes (B, C), granulocytes isolated from the blood (D, E), and a control (CO) section (F). (A) Numerous larger and denser granules (arrowhead) and smaller and paler granules (arrows) are present between the two nuclear sections (n), vesicle (v). (B) Immunolabeled dense granule with even and dark background. (C) Labeled dense granule with a reticulate background. (D) Pale granule from a granulocyte isolated from the blood featuring a meshwork of fine filaments over a pale background. (E) Other immunolabeled pale granule showing a fibrous periphery (arrows). (F) Two immunonegative granules (arrows) in a CO section. Scale bars=1 µm (A), 50 nm (B, D, E), 100 nm (C, F).

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  Fig. 4 Ultrastructure of a granulocytes (A) and Ac-BD-27 immunolabeled granules (B, C) within granulocytes of the wound epidermis. (A) Elongated granulocyte (gr) infiltrated between two keratinocytes of the wound epithelium (ke; nu, nuclei). (B) Detail of immunolabeled dense granules. (C) Two unusual types of low labeled heterogenous granules (es, external space of the granule; ec, electron-dense core). Scale bars=2 µm (A), 100 nm (B), 250 nm (C).

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  Fig. 5 Ac-BD-27 immunolabeled granulocytes present in the regenerating epidermis of the tail (ke, keratinocytes). (A) Most gold particles are diffuse over the dense granules (gr) while the cytoplasm is negative. (B) Detail on a labeled dense but heterogenous granule (gr) near the nucleus (nu), possibly in a stage of discharging its content. Scale bars=150 µm (A, B).

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