Abstract

Cetaceans, especially those living in seawater, demonstrate a variety of ill-understood integumentary lesions. Establishing a panel of antibodies that label different immune cells would facilitate reaching definitive diagnoses, and thus treatment and prevention. Studies on intradermal and mucosal DNA mediated vaccines in other species show promising results and indicate that dendritic cells, especially Langerhans cells, are responsible for inducing a protective naive and memory, cellular and humoral immune response. The conservation of Langerhans cells among the mammalian, avian, reptilian, amphibian and fish species studied thus far confirms its importance as one of the most powerful antigen capturing and presenting cells and suggests it should be present in cetaceans, serving an analogous function. These aspects prompted an immunocytochemical investigation, using available species- and nonspecies-specific antibodies, to identify which immune cells, specifically whether Langerhans cells, are present in normal skin from Tursiops truncatus.

Opportunistic skin samples were obtained from living and euthanatized adult and neonate T. truncatus and from an adult human abdominoplasty patient; dolphin lymph node, spleen and thymus samples were obtained from an aforementioned euthanatized adult T. truncatus. Samples were chemically- and/or cryo-preserved for subsequent immunocytochemical investigation using light, fluorescent, confocal and electron microscopy. To promote sensitivity, cryostat sections were processed by a three-step immunocytochemistry protocol, using biotin-streptavidin-diaminobenzidine (DAB) with nickel enhancement, and/or a two-step immunofluorescence protocol, using a FITC complex. For a negative control, the primary antibody was replaced by the corresponding monoclonal purified anti-mouse IgG or monoclonal supernatant anti-human CD4 (OKT4). For a positive control, human skin was labeled with anti-human cytokeratin (AE1/AE3) or anti-human CD1a (BL 6), or later, dolphin skin was labeled with anti-human MHCII (Q 5/13). The experimental panel of primary antibodies and corresponding published species-specific cellular specificity, with particular reference to skin distribution, are shown in Table 1. Those antibodies denoted by bold face or italic conclusively demonstrated positive results in dolphin skin and the other immune organs or only in the other immune organs, respectively.

The most consistent and strongest results were obtained with anti-human and anti-bovine MHCII. The MHCII epitope is present on Langerhans cells as well as several other immune cells. Consequently, immunoelectron microscopy is being conducted to identify the MHCII antibody-labeled skin cells according to ultrastructure, specifically looking for the Birbeck granule that characterizes Langerhans cells. Considering the biochemical and structural differences in cetacean skin from their terrestrial counterparts, the distribution and morphology of most MHCII antibody-labeled cells in the dolphin skin is consistent with findings of Langerhans cells in previously studied species. Specifically, the labeled cells line the borders of dermal papilla, often sending extensions into the epidermal stratum germinativum, and occur sporadically in the epidermal stratum spinosum. The distribution and morphology of antibody-labeled cells in the lymph node, thymus and spleen support this interpretation. Identifying and possessing a marker for these potent antigen presenting cells, as well as other immune cells, in cetacean skin should facilitate the development of efficacious DNA mediated vaccines and the prevention, diagnosis and treatment of various integumentary lesions.

Table 1. Panel of primary antibodies and corresponding species-specific cellular specificity, with particular reference to the integument.

a. Allotype variant of human CD1a.
b. CC40 is slightly different in cellular distribution than CC14, CC20.
c. No direct study of CD11b distribution in bovine skin, and results from studies in other species differ.
d. No homologue in humans; IL-A24 is a different epitope than CC149.
Paranthesis indicate lack of information in the target species for the specified antibody distribution, but expected results based on studies with other clones in the specified species or in other species.
Bold-face or italic lettering indicate the antibody conclusively demonstrated positive results in dolphin skin and other immune organs or only other immune organs, respectively.
m/s and m/p denote monoclonal/ supernatant and purified antibody, respectively.
Bo = bovine; Ca = canine; Ce = cetacean; Fe = feline; Hu = Human.