The skin contributes to the immune system by acting not only as a protective physical barrier, but also as a target for immune components that mount the initial defense against invading pathogens, noxious stimuli, and resident neoplastic cells. The skin of dolphins, porpoises, and whales (Order Cetacea) has characteristics that distinguish it from that of terrestrial mammals. Such differences are not surprising, considering cetaceans left the land approximately 55 million years ago to adapt to a solely aquatic habitat (Gingerich et al., 1983). Defining characteristics of cetacean skin include lipokeratinocytes, epidermal thickness, a high cell turnover rate, and deeply invaginating dermal papillae (Simpson and Gardner, 1972; Harrison and Thurley, 1974; King, 1974; Brown et al., 1983; Geraci et al., 1986; Menon et al., 1986). It has been proposed that the latter features, and perhaps the resident microflora and antimicrobial properties of the intercellular lipid, contribute to its immunological function (Geraci et al., 1986).
Several skin diseases and immune and reparative responses have been described for cetaceans; however, the etiology and significance of many other lesions remain unclear (Simpson and Gardner, 1972; Harrison and Thurley, 1974; Geraci et al., 1986; Tarpley, 1987; Ridgway et al., 1998; Wilson et al., 1999). The cetacean immune system in general is poorly understood, but it has been the subject of many investigations in recent years (Ness et al., 1998; Romano et al., 1999; Aldridge et al., 2001 [review]; Inoue et al., 2001; Shoji et al., 2001; De Guise et al., 2002; Lundqvist et al., 2002; Romano et al., 2002; Shiraishi et al., 2002). Despite advances in our knowledge of this system, however, little is known about immune cells that may populate the skin, and very few cetacean-specific immunoreagents are available for investigative purposes (Geraci et al., 1986; Romano et al., 1992, 1993; Kumar and Cowan, 1994; De Guise et al., 1997; Romano et al., 1999; Beinke et al., 2001; Aldridge et al., 2001; De Guise et al., 2002).
In most species, the primary antigen presenting cells (APCs) in the integument are the epidermal Langerhans cells and dermal dendritic cells, which have not been reported in cetacean skin. These cells are members of a heterogeneous, dynamic population of APCs, collectively known as dendritic APCs, that are located in discrete areas of nonlymphoid and lymphoid organs, creating an immunological network with defined pathways for movement and homing (Lotz and Thomson, 2001; Girolomoni et al., 2002; Stoitzner et al., 2002). Their function and peripheral distribution make Langerhans cells particularly important as sentinels, messengers, and adjuvants of the immune system. Langerhans cells have been identified in all other species studied thus far by researchers using a variety of techniques, including species-specific and crossreactive immunolabeling (Parsons and MacHugh, 1991; Romano and Balaguer, 1991; Saint-Andre Marchal et al., 1995, 1997; Lotz and Thomson, 2001; Perez-Torres and Ustarroz-Cano, 2001)
In this study, we endeavored to define the presence and distribution of dendritic APCs (especially Langerhans cells) in dolphin skin, using immunohistochemistry. Diseased skin was also examined, since dendritic APCs may be more numerous and their cell surface receptors may be up-regulated (Lotz and Thomson, 2001). Given the lack of dolphin-specific reagents, antibodies were tested that are specific for Langerhans and dermal dendritic cells in other species (primarily those for ungulates and carnivores). Antibodies also were tested in dolphin lymph node, spleen, and thymus to help evaluate the accuracy of crossreactions in skin. This methodology was chosen for its potential and practical applications to cetacean clinical medicine and pathology.
MATERIALS AND METHODS
Tissue Collection and Preservation
Skin was collected opportunistically from adult male bottlenose dolphins (T. truncatus), designated as SNA, BUG, and BUS, during clinical procedures in conjunction with the U.S. Navy Marine Mammal Program. From SNA, a 6-mm punch biopsy was taken immediately left of the dorsal fin after topical analgesia. Using the same preparation and location, a clamp biopsy was taken from BUG. From BUS, 4-mm and 8-mm punch biopsies were taken from the ventrum, caudal to the pectoral flippers. All procedures followed a protocol approved by the Institutional Animal Care and Use Committee under the guidelines of the Association for the Accreditation of Laboratory Animal Care. Skin specimens were also collected during necropsies conducted by the U.S. Navy Marine Mammal Program or the Texas Marine Mammal Stranding Network on dolphins designated as YOG and PO372. YOG, an adult male, died from complications secondary to gastrointestinal disease. PO372, a subadult male, stranded and was euthanized due to progressive meningoencephalitis. BUG, SNA, YOG, and PO372 exhibited no significant dermatopathology, while BUS had dermatopathology of unknown etiology. The spleen, thymus, and mesenteric lymph node were also collected from PO372 to evaluate specificity of antibodies. Human skin was collected opportunistically from an adult female during a cosmetic abdominoplasty to serve as a positive control for the human antibodies tested.
The collected samples were cut into 1-mm-thick blocks and snap-frozen in liquid nitrogen or fixed in 4% paraformaldehyde containing 0.1 M sodium phosphate buffer, pH 7.2. After 24 hr in fixative, the tissue blocks were transferred to 0.1 M sodium phosphate buffer containing 30% sucrose for 48 hr, subsequently frozen on dry ice, and stored in liquid nitrogen until further processing.
The antibodies tested, and the specificity, source, isotype, and concentrations used on respective organs with different immunostaining techniques are summarized in Table 1. Antibodies against bovine, canine, feline, and human CD1 (a–c) family antigens, which label Langerhans and dermal dendritic cells, were tested on dolphin skin from PO372 and YOG for crossreactivity. Q5/13, an antibody to human major histocompatability complex class II (MHC II) molecules (HLA-DR), which has been shown to recognize MHC II on circulating dolphin lymphocytes (Romano et al., 1992), was used to label MHC II (+) cells in skin from each of the dolphins. Cetacean-specific T and B lymphocyte antibodies were used to identify lymphocytes in dolphin skin from PO372 and BUS. Human skin was used as a positive control for the human antibodies tested (BL6 and Q5/13), and the thymus, spleen, and lymph node from PO372 were used to assess the specificity of all antibodies tested. Mouse isotype IgG antibodies or negative monoclonal supernatant were used as negative controls. Secondary antibodies were biotinylated or conjugated with fluorescein isothiocyante affinity purified F(ab)′2 goat anti-mouse IgG (Beckman Coulter Co., Brea, CA).
Table 1. The antibodies tested to investigate immune cells in dolphin skin
Unlike bovine, human MHCII has been characterized in peripheral blood from T. truncatus (Romano et al., 1992).
No immunoprecipitation data.
Allotype variant of human CD1a.
Likely homologue of human CD1b, but different epitope of bovine (Howard et al., 1993).
Frozen tissue blocks were embedded in O.C.T. (Tissue-Tek®; Sakura Fintek Inc., Torrance, CA). Then, 5-μm-thick sections were cut on a cryostat (minotome plus; IEC, Needham Heights, MA) and lifted onto glass slides (Superfrost plus; Fischer Scientific, Tustin, CA). Tissue sections were dried at room temperature for 12–24 hr. Tissues not previously fixed in 4% paraformaldehyde were placed in cold acetone for 5 min and subsequently dried at room temperature. Sections were encircled with a pap pen (Kiyota International, Inc., Rolling Meadows, IL) and dried for 30 min. Slides were rinsed in 0.15 M sodium phosphate buffer, pH 7.2, and incubated for 5 min in 2.14% sodium periodate in 0.05 M Tris + 0.6% NaCl buffer, pH 7.2, to inhibit endogenous peroxidase activity. Subsequently, slides were washed 3 × 3 min in 0.15 M sodium phosphate buffer and incubated in 10% normal goat serum in 0.15 M sodium phosphate buffer for 30 min. Sections were incubated with primary antibody (see Table 1) in 0.15 M sodium phosphate buffer for 1 hr in a humidified chamber on ice. Slides were washed 3 × 3 min in 0.15 M sodium phosphate buffer and subsequently incubated with either affinity-purified F(ab′)2 biotinylated goat anti-mouse IgG (H+L) (Beckman Coulter Co., Brea, CA) at 1:1000, or affinity-purified F(ab′)2 fluorescein isothiocyante goat anti-mouse IgG (H+L) (Beckman Coulter Co.) at 1:200, diluted in 0.15 M sodium phosphate buffer + 1% dolphin serum for 30 min in a humidified chamber on ice.
Fluorescein isothiocyante-labeled sections were rinsed in 0.15 M sodium phosphate buffer and coverslipped with Immunofluore anti-fade mounting media (ICN Biomedicals Inc., Costa Mesa, CA). Sections incubated with biotinylated secondary antibody were washed 3 × 3 min in 0.15 M sodium phosphate buffer and incubated in Streptavidin-HRP (Zymed, San Francisco, CA) at 1:750 in 0.15 M sodium phosphate buffer for 30 min on ice. Sections were washed 3 × 3 min in 0.15 M sodium phosphate buffer, followed by 3 × 3 washes in 175 mM Na acetate-10 mM imidazole, pH 7.0, and incubated in a solution containing 0.03%, 3,3 diaminobenzidine tetrahydrochloride + 0.1 M nickel (II) sulfate + 7H2O + 0.003% H2O2 in 125 mM Na acetate-10 mM imidazole buffer, pH 7.0, which stains the reaction product black. Sections were rinsed 3 × 1.5 min in 0.15 M sodium phosphate buffer, dehydrated, and coverslipped for light microscopy. In some instances, sections were counterstained with methylene blue or periodic acid-Schiff (Armed Forces Institute of Pathology, procedure #HS34) before dehydration. All tissues sectioned (dolphin and human skin, and dolphin lymph node, thymus, and spleen) were stained routinely with hematoxylin and eosin (H&E) to help locate staining by immunofluorescence and immunohistochemistry, as well as to review the basic morphology of the dolphin.
Slides were analyzed under a Nikon Microphot-FXA microscope (Tokyo, Japan) with epifluorescent capabilities. Confocal microscopy was used in some instances to improve interpretations of cellular morphology and identification by following cell bodies and/or cytoplasmic processes in three dimensions (results not shown).
The general morphology of dolphin skin has been described previously; however, for the purposes of this study, dolphin and human skin were routinely stained with H&E to revisit their respective morphologies and to determine the location of immune components in the skin. A comparison of the general morphology of dolphin and human skin is shown in Figure 1. Both dolphin and human skin have epidermal and dermal layers, but the architecture, components, and cellular biochemical composition of these layers contrast in several ways (see Discussion for details).
The immunoreactivity of each antibody with human and dolphin tissues using immunohistochemistry and/or immunofluorescence techniques is summarized in Table 1. Results are reported only when positive and negative controls demonstrated appropriate staining characteristics. When both techniques (immunohistochemistry and immunofluorescence) were used with the same tissue and antibody, and with similarly fixed tissue from different animals, the immunoreactions were essentially identical in distribution and intensity. In general, immunofluorescence was used to screen for crossreactivity and to analyze in three dimensions, and immunohistochemistry was used to further localize staining.
In an attempt to exclusively identify Langerhans cells in dolphin skin, tissues were incubated with human, feline, and canine antibodies to CD1a. Human skin was used as a positive control for anti-human CD1a. None of the antibodies crossreacted with dolphin tissue; however, in human skin, Langerhans cells were immunoreactive for anti-human CD1a, as anticipated. Canine, feline, and/or bovine antibodies to CD1b and CD1c were tested for crossreactivity with dolphin dendritic APCs. Anti-bovine Cd1b (CC40) and anti-feline CD1c were immunoreactive in dolphin thymus (Fig. 2), spleen and lymph node (not shown), but did not exhibit immunoreactivity in skin.
Several, more general antibodies were investigated because of the non-crossreactivity of those specific for Langerhans cells and/or dermal dendritic cells. These included antibodies to MHC II, CD18, CD 11b/18, and MyD-1, from several species. The MHC II antibodies showed positive results, whereas all others were negative. Langerhans cells and dermal dendritic cells, as well as macrophages and lymphocytes, express MHC II; thus, dolphin skin was incubated with anti-human and anti-bovine MHC II. The staining of human skin with anti-human MHC II, used as a positive control, showed a similar distribution to that of CD1a in the epidermis, as well as additional staining in the dermis with a perivascular concentration, as expected (Fig. 3). Both human and bovine MHC II antibodies crossreacted and labeled cells with the same morphology and distribution in dolphin skin. However, anti-human MHC II was preferentially further investigated because it was previously shown to immunoprecipitate radiolabeled detergent lysates of dolphin peripheral blood lymphocytes (Romano et al., 1992).
In dolphin skin, MHC II (+) cells predominated in dermal papillae, primarily located along the epidermal–dermal junction (Fig. 4). It was difficult to discern which layer contained these cell bodies; thus, periodic acid-Schiff counterstaining was used to outline the epidermal basement membrane. Accordingly, these cells were located in the dermis. Cell bodies were polyhedral to triangular, with irregular, elaborate cytoplasmic processes forming a delicate network that reached laterally and occasionally into the epidermis. In addition, MHC II (+) cells were observed scattered in the deeper dermis, often with a perivascular concentration. These cells were either similar in morphology to those in the dermal papillae, or rounder, with less extensive processes. Rarely, the former were also observed in the middle to deep stratum (s.) spinosum. In some sections, only the cytoplasmic processes were apparent, and these might have been misinterpreted as artifact if the tissues had not been examined by 5-μm serial sectioning (Fig. 5) or confocal microscopy (not shown). This problem was identified in human skin as well (de Jong et al., 1986).
Cetacean-specific antibodies to T (anti-CD2) and B (anti-CD21) lymphocytes showed no positive staining in healthy dolphin skin (not shown), although they were positive (as expected) in the thymus, spleen, and lymph node (Fig. 6). The lack of CD2 (+) and CD21 (+) cells in dolphin skin precludes their inclusion among the MHC II (+) cells, and is consistent with the dendritic morphology of the MHC II (+) cells.
Lastly, antibodies were tested on diseased dolphin skin because of the potential increase in dendritic cells and up-regulation of their cell surface receptors. Diseased skin (Fig. 7) was taken from the leading edge of a linear ulceration, as defined by effacement of the basement membrane, including that of the dermal papillae. This lesion was part of a chronic, intermittent dermatologic condition that was confined to the dolphin's ventrum. Grossly, there was marked, diffuse erythema with extensive, eroded to ulcerated nodules that progressed to linear ulcerations. Histologically, ulcerated areas were infiltrated by neutrophils and fewer macrophages and lymphocytes, with multiple hemorrhagic foci. The etiology could not be identified, even after the use of special stains and the available immunohistochemistry techniques (including Gram, Gomori's methenamine silver, periodic acid-Schiff, s-100, and their appropriate controls).
In similar diseased areas of skin, as well as in more normal regions, no crossreaction was seen with the antibodies to CD1a, CD1b, CD1c, CD18, or MyD-1. However, there appeared to be an increase in MHC II (+) cells immediately adjacent to and over the ulcerated area (Fig. 7c). In particular, the cells with elaborate dendritic-like processes were concentrated at the epidermal–dermal junction at the tips of dermal papillae and in the adjacent epidermis. Moreover, there were no CD21 (+) cells (data not shown) and only a few CD2 (+) cells (Fig. 8). The latter cells stained weakly and were located primarily in the superficial region of dermal papillae deep to the ulcerated skin. These findings agree with the histopathology of a predominantly neutrophilic infiltrate.
The skin is the first line of defense against potential pathogens, noxious stimuli, and resident neoplastic cells, and contains cells that interact to form skin-associated lymphoid tissues (SALT). Dendritic APCs (i.e., epidermal Langerhans cells and dermal dendritic cells) constitute a critical component of SALT. Langerhans cells have a regular distribution, dendritic processes, migratory capacity, and dynamic array of antigen receptors, enzymes, and adhesion molecules that respond to changes in the microenvironment. These characteristics are critical for cutaneous immunosurveillance against epicutaneous and intracutaneous insults, and activity in several disease processes, as well as for responses to immunotherapeutics and potential induction of immunological tolerance (Lotz and Thomson, 2001; Numahara et al., 2001; Girolomoni et al., 2002; Stoitzner et al., 2002). Therefore, we investigated the presence of immune cells, focusing on Langerhans cells, in dolphin skin.
Before describing the distribution of MHC II (+) cells in dolphin skin, a general review of the structural and functional morphology of dolphin skin is necessary, given the fundamental differences between it and other mammalian skin (Simpson and Gardner, 1972; Harrison and Thurley, 1974; King, 1974; Brown et al., 1983; Geraci et al., 1986; Elias et al., 1987; Pfeiffer and Jones, 1993; Colbert et al., 1998). For example, the dolphin epidermis is approximately 15–20 times thicker than that of humans (1–4 mm thick, depending on body region and species), and contains deeply invaginating rete pegs that interdigitate with extensive dermal papillae (Brown et al., 1983). There are three epidermal layers: the s. externum (corneum), s. spinosum, and s. germinativum (basale) (Geraci et al., 1986). The s. granulosum and s. lucidum found in human skin are absent. The term “externum” is preferred to “corneum,” because this layer is parakeratinized rather than keratinized. The s. spinosum, the thickest layer, is comprised of interdigitating cells that migrate in a nonuniform manner from their origin in the s. germinativum. The s. germinativum layer is a single cell thick and borders the dermis, as in human skin. Lipokeratinocytes are the basic cellular components of the dolphin epidermis. Their different biochemical composition indicates there are functional differences between lipokeratinocytes and the keratinocytes of human skin (Elias et al., 1987). The relatively thin dermis of dolphin skin has a collagen stroma similar to that of humans, but generally lacks adnexal structures and is more richly impregnated with elastic fibers and nerve fascicles, with a gradual transition into the hypodermis. In addition, the extensive dermal papillae carry an intricate array of lymph and blood vessels and nerve bundles deep into the epidermis.
These morphologic variations, as well as differences in environmental challenges, between dolphins and terrestrial mammals may help explain the slightly modified distribution of MHC II (+) cells found in dolphin skin. The MHC II (+) cells that lie predominantly within the dermal papillae along the epidermal–dermal junction in dolphins exhibit a dendritic-like morphology and network arrangement similar to that of the Langerhans cells that occupy a suprabasalar position in terrestrial mammals. Such features suggest that they are more likely Langerhans cells than dermal dendritic cells or macrophages. The latter two cells probably represent the MHC II (+) cells found in the deeper dermis. These cells either had a morphology similar to the former cells, or a round cell body with less extensive cytoplasmic processes. In addition, they exhibited a scattered distribution with a perivascular concentration, which is similar to that observed in terrestrial mammals (Lotz and Thomson, 2001). Finally, T and B lymphocytes were not among the MHC II (+) cells in healthy dolphin skin, as determined by immunostaining with cetacean-specific CD2 and CD21, respectively.
This tentative cell identification is not only consistent with the morphology, distribution, and immunophenotype of the respective immune cells, but also with dolphin skin morphology, response to insult, and repair mechanisms. In dolphin wound healing, there is no scab formation (King, 1974). Instead, there is a zone of degenerating spinosum cells that undergo hydropic changes, and subsequently are sloughed and rapidly replaced from a focus of mitosis in the s. germinativum. As repair progresses from deep to superficial epidermis, the buffer zone becomes thinner and eventually disappears. Often inflammation is not present, because this barrier is effective against common benign insults sustained by the thick epidermis, including minor behavioral interactions with other dolphins (i.e., rake marks) and temporary dryness resulting from the dolphin resting on the surface. Thus, with this relatively thick buffer zone, high epidermal cell turnover rate, and extensive array of dermal papillae, the cutaneous immunosurveillance cells (such as Langerhans cells) may be more appropriately poised just deep to the epidermis.
When inflammation is incited, as demonstrated by the ulcerated skin used in this study, immune cells apparently migrate superficially from the underlying dermal papillae toward the insult. The diseased skin (of unknown etiology) used in this study had an inflammatory infiltrate comprised predominantly of degenerate and nondegenerate neutrophils, and fewer macrophages and lymphocytes. At the leading edge of the lesion, H&E staining showed a minimal inflammatory infiltrate, especially in the epidermis. Immunohistochemistry, however, showed an increased population of MHC II (+) cells that had an elaborate dendritic morphology. These cells were concentrated in the superficial dermis and adjacent epidermis of dermal papillae. Considering the lack of immunostaining for B lymphocytes with cetacean-specific CD21, minimal immunostaining for T lymphocytes with cetacean-specific CD2, and suppurative nature of the inflammatory infiltrate, the increased population of MHC II (+) cells most likely represents Langerhans cells or dermal dendritic cells. In other species, Langerhans cells have been shown to migrate toward chemical and neurological stimuli to induce or sustain naïve cellular, humoral, and memory immune responses (Miyan et al., 1998; Scholzen et al., 1998; Lotz and Thomson, 2001).
A definitive identification of the MCH II (+) cells in dolphin skin as Langerhans cells, dermal dendritic cells, or macrophages will require confirmation with more specific antibodies, molecular techniques, and/or ultrastructural analysis by electron microscopy. Langerhans cells contain a unique ultrastructural component known as the birbeck granule (Lotz and Thomson, 2001). This is considered an important criterion for positive identification of Langerhans cells, although its precise function remains speculative (Valladeau et al., 2000). Further antibody and ultrastructural studies for definitive identification are under way. Future studies will be carried out on a larger sample size, and include comparisons of skin samples obtained from different anatomical locations, different ages and genders, and ante- and postmortem samples (including some from defined disease processes).
In this study, immunoreactivity of one bovine CD1b and one feline CD1c antibody in dolphin thymus, spleen, and lymph node was consistent with that found in other species relative to cetacean histology (Romano et al., 1993). However, neither antibody crossreacted in healthy or diseased dolphin skin. Interestingly, of the three anti-bovine CD1b antibodies tested, only CC40 crossreacted with dolphin. This antibody has a slightly different distribution in bovine tissue than CC14 and CC20, and may not be a true homologue of CD1b (Parsons et al., 1991; Howard et al., 1993; Howard and Naessens, 1993). Additionally, in species-specific reactions, CD1b shows weak staining for dermal dendritic cells in healthy skin, and additional staining for epidermal dendritic cells in diseased skin of various etiologies. Thus, the absence of staining in healthy dolphin skin was not surprising, but was not expected in diseased dolphin skin. This discrepancy may reflect labeling of a similar but different epitope in the thymus, spleen, and lymph node. A similar explanation may apply to the staining pattern observed with anti-feline CD1c. More specifically, CD1c shows variable staining of epidermal dendritic cells among species, but consistent and strong staining of dermal dendritic cells in all species studied thus far. Further investigation is required to assess the significance of these findings.
We have identified MCH II (+) cells with a dendritic-like morphology in the skin of T. truncatus. Based on morphology, distribution, and immunophenotyping, these cells may be Langerhans cells in the superficial dermis, and dermal dendritic cells and/or macrophages in the deeper dermis. The former cells may assume a location different from that in terrestrial mammals due to differences in dolphin skin composition, morphology, and function, and in environmental challenges. Moreover, in states of disease, dendritic-like cells may up-regulate MHC II and migrate into superficial layers. Further immunological characterization of cetacean skin using immunohistochemisty techniques will enhance our knowledge about the evolution of functional adaptations to different environments, and facilitate the diagnosis of skin diseases. Finally, an understanding of cetacean skin immunology will define the potential for intradermal administration of vaccines and immunotherapeutic agents.
The authors are indebted to Dr. Steven E. Poet for his invaluable contributions, and to Dr. Walter L. Steffens and Mary B. Ard for their help with the electron microscopy. The authors thank Dr. Daniel F. Cowan, the U.S. Navy Marine Mammal Program, and the Center for Plastic Surgery and Reconstruction for the tissues, and Drs. Chris J. Howard, Peter F. Moore, George F. Murphy, Vito Quaranta, and Jeff L. Stott, and the Beckman Coulter and Boehringer Mannheim Corporations for donating the antibodies. The authors thank Dr. Chris Dascher for the photomicrograph of human skin, and for reviewing this manuscript. The authors also thank Drs. Branson W. Ritchie, W. George Miller, William Van Bonn, Elizabeth W. Howard, Pauline M. Rakich, and Wes Hall for their expertise and assistance, as well as Chris Write and Esi Djan for technical assistance. This study was supported by grants from the Office of Naval Research (N66001-9216-3507 to T.S. Zabka, and N00014-00-1-0041 to T.A. Romano).