INTRODUCTION
The mucosal surfaces of teleosts (bony fishes) are the major interface between fishes and their immediate environment and serve as primary sites of entry for most pathogens. The mucosal surfaces of fishes include the epithelia and associated tissues of the gills, skin, gut, and the reproductive tract.
In mammals, the mucosal system consists of an integrated network of tissues with associated immune cells referred to as the mucosa-associated lymphoid tissue (MALT). It is generally accepted that a comparable system exists in teleosts, although much less is known about its cellular and molecular components and the extent to which they function independently from the systemic immune response.
Although a general understanding of the teleost immune system is emerging, fundamental questions still remain regarding primary lymphoid organ development, the induction, amplification and differentiation of local mucosal immune responses, the production of mucosal antibodies and effector lymphocytes, and immune memory. Answers to these questions will lead to a greater understanding of the evolution of basic immunological mechanisms as well as insights of immediate relevance to applied vaccines and the protection of farm-reared fish from microbial infections.
A number of laboratories have been or are currently engaged in research on mucosal immunity in various fishes, including, carp (Cyprinus carpio) (Rombout et al., 1993), channel catfish (Ictalurus punctatus) (Lobb, 1987; Hebert et al., 2002), rainbow trout (Oncorhynchus mykiss) (Bromage, 2004), Atlantic salmon (Salmo salar) (Lin et al., 1998), sea bass (Dicentrarchus labrax) (Picchietti et al., 1997), zebrafish (Danio rerio) (Danilova and Steiner, 2002) and others because teleosts are a diverse group of fishes, an understanding of the biology of their immune system requires a comparative approach. From the synthesis of research from various laboratories on multiple fish species, a general understanding of mucosal immunity exists and these concepts are presented in each of the chapter sections.
ORGANIZATION OF MUCOSAL TISSUES AND ASSOCIATED IMMUNE CELLS
Gastrointestinal Tract (Fig. 1.1)
Fig. 1.1 Intestinal epithelium
A. Diagram of the basic anatomical structures of the intestinal epithelium and the identification and location of immune-related associated cells. B. Photomicrograph of the intestinal villus of a channel catfish. Note the mucosal brush border, tall columnar epithelial cells (enterocytes), and supporting lamina propria containing migrating lymphocytes and coarse eosinophilic granulocytes (hematoxylin and eosin [H & E] stain).
The respiratory and digestive systems share the mouth and buccal cavity. The lining of the buccal cavity consists of a stratified mucoid epithelium on a thick basement membrane with a dermis that connects the epithelium to the underlying bone or muscle tissues (Roberts, 2001).
The esophagus has an epithelial lining with large numbers of mucus cells. The stomach varies in size, depending on the species of the fish under study. The gastric mucosa is mucoid with numerous glands in the crypts of the mucosal folds (Roberts, 2001).
Although the intestinal morphology of teleosts varies depending on the species and diet, the intestinal tract has a common basic structure. The intestine is a single tube without the anatomically distinct colon found in mammals (Roberts, 2001).
The rectum has a thicker muscle wall than the intestine and is very mucogenic (Roberts, 2001). The esophagus, stomach, and intestine have four basic layers that vary in composition among and within each of these organs. The innermost layer is the mucosa, which is composed of epithelium, a lamina propria of fibrous connective tissue, and sometimes a muscularis mucosae.
The submucosa, comprised of fibrous connective tissue, lies between the mucosa and the muscularis, which is completely made of muscle. The outer layer of the serosa is composed of fibrous connective tissue covered with a simple squamous mesothelium (Grizzle and Rogers, 1976). The intestinal mucosa is considered to be an immunologically important site in teleosts (Cain et al., 2000).
In carp, the posterior segment of the gut, referred to as the second gut segment, plays a significant role in mucosal immunity (Rombout and van den Berg 1989; Rombout et al., 1989; Rombout et al., 1989) and comprises 20-25% the length of the gut (Rombout et al., 1993; Press and Evensen, 1999). The gut-associated lymphoid tissue of most teleosts, including rainbow trout (McMillan and Secombes, 1997), carp (Rombout et al., 1993), and sea bass (Picchietti et al., 1997) is comprised of cells with lymphoid morphology residing between the gut epithelial cells.
These are predominantly intraepithelial T lymphocytes (Bernard et al., 2006; Huttenhuis et al., 2006), but Ig lymphocytes are also found with the predominant number residing in the lamina propria (Rombout et al., 1993; Danilova and Steiner, 2002; Huttenhuis et al., 2006).
Lymphoid aggregations that resemble the ileal or Peyer's patches in mammals are absent. The GALT of teleosts principally consists of lymphocytes of various sizes, plasma cells, macrophages as well as different types of granulocytes (Du Pasquier and Litman, 2000).
Periodic acid Schiff (PAS) positive cells and eosinophilic granular cells are present, and may serve to modulate immune-hypersensitive responses that occur in the gut. In the intestinal epithelium and lamina propria, macrophages function as scavengers and antigen presenters.
In carp, intestinal macrophages are different from the macrophages isolated from other lymphoid organs in the sense that they adhere poorly to glass and plastic, form clusters with lymphocytes, express antigenic determinants on their outer membranes and bind immunoglobulin (Ig) (Rombout et al., 1986, 1989 a, b, 1993).
The biliary system of the liver begins with intracellular bile canaliculi that anastamose extracellularly to form bile ducts. These fuse into the gall bladder, which directs bile into the intestine through the common bile duct. The gall bladder is lined with transitional epithelium. Hematopoietic tissue with melanomacrophage centers is associated with larger blood vessels of the liver (Roberts, 2001).
Skin (Fig. 1.2)
Fig. 1.2 Skin epithelium
A. Diagram of the basic anatomical structures of the skin and the identification and location of immune-related associated cells. B. Photomicrograph of channel catfish skin (sensory barbel) (H & E stain).
The skin of fishes provides protection against physical, chemical and biological damage. It consists of two anatomical layers, the epidermis and dermis. The thickness of the stratified epithelium of the epidermis varies, depending on the area of the body, age, sex, maturation and environmental stresses (Grizzle and Rogers, 1976; Yasutake and Wales, 1983). On average, it has a thickness of 10-12 cells.
Cells in the basal columnar layer of the epidermis, referred to as the stratum germinativum, replicate and move toward the surface of the fish. This basal layer lies immediately above a basement membrane. At least six types of cells have been described in the epidermis of teleosts, including filament-containing malpighian cells (keratinocytes), mucus cells, chemosensory cells, club cells (alarm substance cells), granule cells and chloride cells (Grizzle and Rogers, 1976; Yasutake and Wales, 1983; see for review zaccone et al. 2001).
The malpighian cells are the most abundant in the epithelium. These cells are rounded in shape with bundles of fibers and mitochondria around a generally ovoid nucleus (Roberts, 2001). At the epithelial surface, keratinocytes become more flattened and their cytoplasm consisting predominantly of oblong vesicles, degenerating mitochondria and denser bundles of fibers. The outermost layer of cells is not keratinised.
The surfaces of the outermost cells have convoluted microridges of an unknown function that possibly assist in holding mucous secretions to the skin. Mucus cells begin differentiating in the stratum germinativum and migrate to the surface of the skin where they release their contents. Packets of mucus are bound by membranes and progressively fill the cell as they move toward the surface. At the surface of the epithelium, the mucus cell (a holocrine gland) moves between the keratinocytes and discharges its contents. The epidermis is covered by a glycocalyx or cuticle, consisting of a thin (1.0 µm) mucopolysaccharide layer. It is a complex mixture of molecules derived primarily from the contents of sloughed surface epithelial cells and mucus secreted from goblet cells (Roberts, 2001).
The deeper layers of the epidermis contain alarm substance cells and melanophores, which do not reach the surface. The contents of alarm substance cells are only released when the epidermis is physically damaged (Grizzle and Rogers, 1976), Capillaries extend into the epidermis from dermal papilli, and come within 10 cell layers of the surface (Lobb, 1987). The dermis is composed of two layers. The upper layer, referred to as the stratum spongiosum, consists of a loose network of collagen and reticulum fibers and is contiguous with the epidermal basement membrane that lies just above it. It contains chromatophores, mast cells and the cells of the scale beds. The lower layer, the stratum compactum, is a dense matrix of collagen that provides the structural strength to the skin. The hypodermis, lying beneath the dermis, is composed of loose connective tissue. It is more vascular than the overlying dermis. Melanophores occur in the hypodermis, dermis and sometimes in the epidermis. No organised lymphoid germinal centers have been found in the skin (Flajnik, 1998), although cells with the morphology of lymphocytes can be detected by light microscopy in stained tissue sections of channel catfish skin (Lobb, 1987).
These cells occur throughout the epidermis and are located predominantly near the stratum germinativum at the junction of the epidermis and dermis (Lobb, 1987). Antigen-specific and total antibody secreting cells (ASC) have been isolated from the skin of channel catfish and detected by ELISPOT (Zhao et al., 2007). B cells isolated from the skin of channel catfish can be stimulated with LPS to replicate and secrete antibody in vitro, a response that, in turn, is abrogated by the addition of hydroxyurea to the culture medium (Zhao et al., 2007). Macrophages are also present in the skin (Roberts, 2001).
Gills (Fig. 1.3)
Fig. 1.3 Gill epithelium
A. Diagram of the basic anatomical structures of the gill epithelium and the identification and location of associated immune-related cells. B. Photomicrograph of fish gill. Note the capillaries with erythrocytes in secondary lamellae and chloride cells concentrated in lamellar troughs (H & E stain).
The gills consist of gill arches, gill filaments (primary lamellae), and gill lamellae (secondary lamellae). Two rows of filaments are present on each arch and the secondary lamellae branch out perpendicularly from the filaments (Grizzle and Rogers, 1976; Yasutake and Wales, 1983). The gill arches and filaments are supported by a branching system of cartilaginous rods. A stratified squamous epithelium covers both the gill filament and the gill lamellae. The lamellae provide the actual respiratory surfaces.
Each lamella comprises a network of interconnected spaces that are separated and supported by pillar cells. Blood enters the lamellae from the afferent arterioles of the filaments and exits into the efferent arteriole. The lamellar intercellular spaces through which blood flows are lined with endothelial cells. A basement membrane lies over the endothelial cells and pillar cells, which form supportive 'flanges' around the intra-lamellar spaces (Grizzle and Rogers, 1976). The stratified epithelium itself is only one to two cells thick in order to allow gas exchange, a degree of thinness that makes the tissue vulnerable to invasion by pathogens. Different cell types are associated with the gill epithelium. Chloride cells function in the transport of Cl and other ions across the epithelium. These cells are more spherical than those that surround them in the epithelium; they project somewhat above the surface (Yasutake and Wales, 1983), and their cytoplasm is more eosinophilic (in hematoxylin and eosin stained sections) than is the case with other epithelial cells. Chloride cells are abundant in the gill filament epithelium between lamellae (Grizzle and Rogers, 1976). Mucus cells are abundant in the lamellar epithelium, and appear under light microscopy as mucus-filled domes or vacuolated cells (Yasutake and Wales, 1983). Goblet cells are most abundant on the margins near arterioles. Alarm substance cells are absent in gill epithelia (Grizzle and Rogers, 1976). Although the surface of the gill lamellar epithelium is irregular, it does not have the distinctive microridges seen on the surface of the skin epidermis (Roberts, 2001). Nevertheless, these irregularities are sufficient to aid in attachment of mucus, which in addition to its role in reducing invasion of microorganisms, also serves to regulate the transfusion of gases, ions, and water across the epithelial membrane (Roberts, 2001). Similar to the situation that exists in skin, there is no evidence to indicate the existence of organised aggregations of lymphoid tissue in the gills. Nevertheless, there have been a number of studies to show the functional immunological activity in gills as well as gill-associated leucocytes and lymphocytes (Goldes et al., 1986; Powell et al., 1990; Lumsden et al., 1995; Davidson et al., 1997; Lin et al., 1998; Rombout et al., 1998; Dos Santos et al., 2001 a,b). Considerable numbers of lymphocytes, ASC, and macrophages were found to reside in the gill tissue of Atlantic salmon and dab (Lin et al., 1998). In leucocyte suspensions from carp gill (as in skin), Rombout et al. (1998) found an abundant population of intraepithelial lymphocytes (IEL) that reacted with a monoclonal antibody (mAb WCL38), which is specific for IEL T cells in the carp intestine. In gill IEL leucocyte suspensions, WCL38+ cells comprised the major population of lymphoid cells. Lymphocytes with surface immunoglobulin (i.e., B cells) were a minor component of these cell populations. In cryosections, many of the WCL38+ cells were detected at the base of the gill lamellae. Immunogold labeling showed that the WCL38+ cells had the ultrastructure of lymphoid cells, although two morphologically different cell types were found: small lymphocytes with a high nucleus/cytoplasm ration, and larger granular lymphocytes with a lower nucleus cytoplasm ration and a variable amount of electron-dense, lysosome-like material.