Mucosal immunoglobulins and B cells of teleost fish(진행중)
1. Introduction
Higher metazoans have different barriers that separate themselves from the surrounding environment. Whereas some animal species evolved non-mucosal barriers (i.e. the cuticle of arthropods), others, such as teleost fish, developed mucosal surfaces as their strategy to protect themselves from the aggressions of the environment.
In addition to being physical barriers, mucosal surfaces are also active immunological sites armed with cellular and humoral defences. Since these surfaces represent the interface between each animal and the external environment, they are exposed more than any other site, to a continuous bombardment of microbes and stressors.
The mucosa-associated lymphoid tissue (MALT) contains B cells and immunoglobulins, which play a pivotal role in the maintenance of mucosal homeostasis (reviewed by Brandtzaeg, 2009). In higher vertebrates, secretory immunoglobulins (sIg) as well as their importance in innate and adaptive immunity, are fairly well characterized. Despite the fact that sIg in mammals has been classically associated with IgA and to a lesser degree with IgM, there is a growing appreciation that all immunoglobulin classes are in fact relevant at mucosal sites (Baker et al., 2010).
In lower vertebrates, and in particular teleost fish, the presence of immunoglobulins in mucosal secretions was first reported in plaice (Pleuronectes platessa) in the late 1960s (Fletcher and Grant, 1969). Until recently, there has been a general belief that IgM was the only functional immunoglobulin in teleosts, both in systemic and mucosal compartments. Recent breakthroughs in the field of fish immunoglobulins have added two new players to the scene, IgD (Edholm et al., 2010a) and IgT/IgZ (Danilova et al., 2005; Hansen et al., 2005).
Significantly, IgT has been reported to be an immunoglobulin specialized in gut mucosal immunity (Zhang et al., 2010), a novel finding that makes the field of mucosal immunoglobulins and mucosal B cells in fish even more appealing. As commented by Flajnik (2010) in reference to recent findings on IgT, all GOD’s (generation of diversity) creatures, even fish!, appear to have dedicated mucosal immunoglobulins. To our delight, there is still much to discover about these immunoglobulins and their immune functions.
The present review summarises our current knowledge on teleost B cells and immunoglobulins found in mucosal surfaces. It also examines, with an evolutionary and comparative eye, the parallelisms and dissimilarities of sIg in bony fish versus higher vertebrates.Moreover, this review attempts to integrate past and current basic and applied research findings of fish mucosal immune responses as a platform to provide new directions that facilitate the future development of novel vaccination strategies. These strategies should target stimulation not only of systemic, but also of mucosal immunity.
2. Gross anatomy of MALT
Obvious physiological, anatomical and histological differences exist between terrestrial and aquatic vertebrates, which clearly translate into the presence of distinct MALT in fish and mammals. It is accepted that the mucosal immune system is more complex than its systemic counterpart both in terms of effectors and anatomy (Brandtzaeg, 2009; Cerutti and Rescigno, 2008; Fagarasan, 2008; Macpherson et al., 2008) and, as a consequence, its nomenclature also becomes more intricate.
The Nomenclature Committee of the Society of Mucosal Immunology proposed a standard nomenclature for both secretory immune-function molecules and mucosa-associated immune-cell compartments. Table 1 includes the nomenclature for mucosa-associated immune-cell compartments accepted in mammals (summarised in Brandtzaeg et al., 2008) as well as those thus far used in fish. It is important to note that no standard nomenclature has yet been proposed for fish MALTs and therefore, we recommend adopting the mammalian one in those cases where it is applicable.
MALT uniquely present in teleosts has its own terminology but unfortunately there has not been a general agreement on it by the scientific community. The three main mucosal immune compartments found in bony fish are: (1) the gut-associated lymphoid tissue (GALT) with the lamina propria (LP) and intraepithelial (IEL) compartments; (2) the skinassociated lymphoid tissue (SALT); (3) the gill-associated lymphoid tissue (we propose to abbreviate it as GIALT) which includes the gills and the interbranchial immune tissue (ILT).
2.1. The gut-associated lymphoid tissue (GALT)
Herbivorous, detritivorous, omnivorous and carnivorous fish species differ from each other in terms of the presence or absence of a stomach, the length of the intestine (from 1 to more than 20 times the body length), and the presence and number of pyloric caeca, intestinal loops and valves (Evans, 1998). The GALT is strikingly diverse across vertebrate groups. For instance, chickens have caecal tonsils not present in mammals, and even within mammals, their GALT exhibit a significant structural diversity (Fagarasan, 2008; Finke and Meier, 2006).
Generally speaking, the GALT of higher vertebrates consists of both scattered and organised lymphoid tissue. Fish, however, lack an organised GALT, and thus, have no peyer’s patches (PP) or mesenteric lymph nodes (MLNs) (Rombout et al., 2010), whereas the presence of PP or MLN in amphibians remains to be demonstrated, although lymphoid accumulations were demonstrated in the lamina propria of the amphibian urodele, Pleurodeles waltlii (Ardavin et al., 1982; Zapata and Amemiya, 2000).
In fish, lymphoid cells are present in a scattered manner along the alimentary canal. The LP and IEL compartments are nevertheless identified. An updated review on the teleost fish GALT, including the description of all the immune cell types therein present has been recently compiled (Rombout et al., 2010) and additional details among different cartilaginous and bony fish are reviewed in Hart et al. (1988) and Zapata and Amemiya (2000).
Generally speaking, teleost gut LP harbours a variety of immune cells including, but not limited to macrophages, granulocytes, lymphocytes and plasma cells, whereas the IEL compartment is mainly composed by T cells and few B cells.
One exception is the halibut (Hippoglossus hippoglossus), where a great diversity of leukocytes is observed in the epithelium of the second segment of the gut (Grove et al., 2006a). Immunological differences are recognised along the different segments of the fish gastrointestinal (GI) tract as it is the case in mammals. Most of our knowledge in that area relates to the differential uptake of particles in the anterior gut (also known as foregut or first segment) compared to the posterior gut (hindgut or second segment). More details about particle uptake in the gut can be found in Rombout et al. (2010). Another example is found in cod (Gadus morhua L.), where clear immunological differences between the second segment of the gut and the rectum exist (Inami et al., 2009).
The geographical map of teleost gut immune cell populations is however far from complete. In that regard, very little is known in particular about the distribution of sIg classes and B cell subsets in different portions of the GI tract. It is worth mentioning that the pH conditions along the fish GI tract change drastically. For instance, catfish (Ictalurus punctatus) has a pH between 2 and 4 in the stomach, then becomes alkaline below the pylorus (pH 7–9). In the foregut the pH is 8.3 and it is near neutral in the hindgut (Pillay and Kutty, 2005).
Thus the question arises, how do pH changes affect antigen uptake, antigen–antibody interactions, or other immune processes? There is still considerable debate regarding which cells are the main antigen collecting cells in the teleost GI tract. Whereas some authors claim that enterocytes uptake certain antigens such as ferritin (Rombout et al., 1985), others suggest the presence of Mlike cells in the posterior gut of salmonids (Fuglem et al., 2010). Some but no conclusive evidence has shown that certain fish GALT epithelial cells display morphological similarities with mammalian M cells and sample luminal antigens (Fuglem et al., 2010).
In cyprinids like carp (Cyprinus carpio L.) (Rombout et al., 1985) and goldfish (Carassius auratus auratus) (Temkin and McMillan, 1986), large intraepithelial macrophages containing phagocytosed material have been observed and therefore these are thought to be the main antigen presenting cells. However, no macrophages were found in the gut of seabream (Sparus aurata) (Mulero et al., 2008). One study measured the respiratory burst response of GALT leukocyte suspensions from the seabream and found very few phagocytic cells (Salinas et al., 2007). It is obvious that we know very little about how antigens are uptaken and presented in fish GALT and once again we can appreciate that the gut of different teleost species is immunologically speaking as variable as their physiology. The microbiota associated to the gut of several teleost species has received a fair amount of attention, mainly due to its application as probiotics in the fish farming industry (Nayak, 2010). It is apparent that commensal microorganisms associated with the gut of fish are more transient and variable in their composition than those of terrestrial animals, probably because of the microbiology of the diverse aquatic environments in which fish live. Initial colonisation of the gut by different microbial species determines GALT early development as well as the total B cell repertoire in different animal models (Fagarasan, 2008; Lanning et al., 2005; Rawls et al., 2004). Moreover, the development of epithelial barrier function and gut innate immunity in gnotobiotic zebrafish appears to be determined by bacterial colonisation in the gut (Rawls et al., 2004). Thus, similar to the situation in higher vertebrates, gut commensal microorganisms are thought to modulate immune responses in the fish gut (Nayak, 2010; Perez et al., 2010; Rawls et al., 2004). In the context of the present review, it is essential to mention the interaction between fish sIg in the gut and their commensal bacteria. Interestingly, IgT and, to a lesser extent, IgM from the gut mucus of rainbow trout (Oncorhynchus mykiss) coat a high percentage of the bacteria present in the gut (Zhang et al., 2010). Thus, bony fish sIg are likely to exert immune exclusion on their commensal bacteria, in a similar manner mammalian sIgA and sIgM do (Brandtzaeg, 2009), thereby providing a means for controlling gut homeostasis.
2.2. The skin-associated lymphoid tissue (SALT)
The SALT (also known as skin immune system (SIS) or cutaneous immune system) in fish is a mucosal lymphoid tissue, whereas in terrestrial vertebrates is not strictly so. Teleost skin is histologically very different to that of mammals since in fish the outermost layer of cells is alive and it retains the capacity to divide. In that regard, teleost epidermis is a stratified but non-keratinized epithelium of variable thickness. Among the epithelial cells, abundant secretory cells are present and produce altogether a complexmucus secretion with ample biological functions. Four types of secretory cells can be found in different fish epidermis including malpighian cells, goblet cells, sacciform cells and club cells (for more details on the structure of fish epidermis refer to the review by Zaccone et al., 2001). Once again, teleost diversity is reflected in a variety of skin morphologies, cell composition, mucus characteristics and molecules present in the mucosal secretions. One example of this diversity is found in the work by Fast et al. (2002), where the skin of three salmonid species was shown to be biochemically and histologically different. Both malpighian and goblet cells have been proposed to be phagocytic in fish skin (Asbakk, 2001; Iger and Abraham, 1990). Moreover, the presence of IgM was detected in the goblet cells of trout skin (Peleteiro and Richards, 1988). In addition to secretory cells, leukocytes such as granulocytes, macrophages and lymphocytes (Davidson et al., 1993a; Herbomel et al., 2001; Iger et al., 1988; Peleteiro and Richards, 1990) have been observed in the skin of different teleost species. Very little information is however available on the function of each cell type during the course of an immune response. The importance of the skin as an immune organ was clearly demonstrated when transcript analysis of common carp revealed 82 orthologues of genes of immune relevance previously described in other organisms (61 of them had never been described before in carp) (Gonzalez et al., 2007). Finally, fish skin mucus harbours an apparently abundant and diverse microbial community including bacteria and fungi (Austin, 2006; Liu et al., 2008). The modulation of SALT by this microbial community is unknown and further studies are clearly required in that area.
2.3. The gill-associated lymphoid tissue (GIALT)
Teleost generally have four pairs of gill arches supported by cartilage or bone tissue. Gill arches contain gill filaments (or primary lamellae), which are subdivided into gill lamellae (or secondary lamellae). These comprise the main respiratory surfaces of the fish. A comprehensive review on fish gill morphology can be found in Wilson and Laurent (2002). Recent morphological studies on nile tilapia (Oreochromis niloticus) have described the branchial filament as a multilayered epithelium, such as in other teleostean species. The filament epithelium, on the other hand, was shown to consist of two distinct regions, a superficial and a deep layer, the latter characterized by a network of undifferentiated cells, wide intercellular spaces where cells from the neuroendocrine and immune systems reside (Monteiro et al., 2010). Small and large lymphocytes (Grove et al., 2006a; Lin et al., 1998), macrophages (Lin et al., 1998; Mulero et al., 2008), neutrophils (Lin et al., 1998), eosinophilic granulocytes (Barnett et al., 1996; Lin et al., 1998; Mulero et al., 2007) and antibody-secreting cells (ASC) (Davidson et al., 1997; dos Santos et al., 2001a) have been observed in the GIALT of different fish species (below in Sections 4.4 and 5.3 we review in more depth GIALT B cells and ASCs). Gill cell suspensions from the dab (Limanda limanda) were exposed to lipopolysaccharide (LPS) and phytohemagglutinin (PHA) and the produced mitogenic responses indicated the presence of few Bcells and a preponderance of T-cells (Lin et al., 1999). Secondary lamellae are formed by a very thin epithelium that is supported by pillar cells. This creates a capillary space for erythrocytes to flow. Thus, it is not common to see lymphoid cells in this area (Monteiro et al., 2010). Many of the anatomical characteristics of interlamellar vessels are strikingly similar to those of mammalian lymphatic capillaries and they have been suggested to be physiologically, if not embryologically, equivalent (Olson, 2002). Interestingly, it has recently been reported that the basal chordate Amphyoxus also possess a gill-associated lymphoid tissue (Han et al., 2010). In addition to the lymphoid tissue found within the gill lamellae, an interbranchial lymphoid tissue (ILT) has been recently described in salmonids (Haugarvoll et al., 2008; Koppang et al., 2010). The organisation of this lymphoid tissue resembles that of the thymus: it is covered by an epithelial layer and traversed by trabecular walls. These studies also showed the predominant presence of T cells in salmon ILT. Therefore, at least salmonid GIALT consists both of dispersed leukocytes within the lamellar epithelium and organised lymphoid areas between gill arches. Mucus production is proven to be higher in the area surrounding the gill cover than in any other skin sites (Shephard, 1994). Additionally, fish gills have an associated microbial community (Ringo and Holzapfel, 2000) which, in the case of the gibel carp (Carassius auratus gibelio) and bluntnose black bream (Megalobrama amblycephala), is less diverse than that of the skin (Wang et al., 2010).
3. Presence and transport of immunoglobulins in mucosal sites
3.1. Teleost serum immunoglobulins
Immunoglobulins are mainly produced by plasmablasts and plasma cells, and are found secreted into body fluids (including serum and mucosal secretions) as antibodies (soluble form), or on the surface of B cells as B cell receptors (BCR) (membrane-bound form). Ig molecules are typically composed of two identical heavy (H) chains and two identical light (L) chains which provide two identical antigen-binding sites by the amino-terminal variable (V) domains of both H and L chains. The H chain carboxyl-terminal constant (C) domains define the Ig isotypes (classes) and the effector functions of the Ig through binding to their receptors on effector cells. The H and L chains are encoded by separate genomic loci, igh and igl respectively, and their V and C domains are each encoded by independent elements: the variable (V), diversity (D, only for H chains), and joining (J) gene segments for the V domain, and individual constant (C) gene segments for the C domains. Up to date, three major Ig isotypes have been reported in teleost fish, among which IgM was the first one discovered decades ago, while IgD and IgT/IgZ were discovered later in 1997 (Wilson et al., 1997) and 2005 (Danilova et al., 2005; Hansen et al., 2005), respectively. Generally, in an igh locus of teleost, for example in rainbow trout, the gene segments (VHDJC) encoding for the H chain () of IgT are located upstream of those (VHDJCCı) encoding for the H chains ( and ) of IgM and IgD (Hansen et al., 2005). In such a locus, the upstream V segments were predicted to rearrange either to DJC to encode chain or to DJC to encode chain, and consequently, B cells of this species were predicted to express either IgT or IgM (Flajnik, 2005). Confirming the aforementioned prediction, in 2010 it was reported that rainbow trout contained a new B lineage uniquely expressing surface IgT, whereas IgM+ B cells were found devoid of IgT expression (Zhang et al., 2010). For further information on the genomic organisation of teleost igh and igl loci, see recent reviews (Edholm et al., 2011; Hikima et al., 2010; Solem and Stenvik, 2006; Sun et al., 2011; Zhang et al., 2011). In general, the prevalent serum Ig in most teleosts is a high molecular weight (HMW) Ig (600–850 kilodaltons (kDa)), corresponding to tetrameric IgM, which is stable under physiological conditions, but under denaturing conditions exists as various redox forms that differ in the degree of inter-heavy chain disulfide polymerization (Bromage et al., 2004b; Kaattari et al., 1998). Recently, an association between the increased disulfide polymerization and the greater affinity of trout IgM to antigen has been reported (Ye et al., 2010). The concentration of teleost serum IgM may vary (0.6–16 mg ml−1) among species andmay change depending on fish size, environment temperature, water quality, season of the year, as well as stress, stimulation, or immunisation (reviewed in Solem and Stenvik, 2006). Besides the HMW Ig, a low molecular weight (LMW) Ig was also described in the serum of giant grouper (Epinephelus itaira) (Clem, 1971), margate (Haemulon album) (Clem and McLean, 1975), sheepshead (Archosargus probatocephalus) (Lobb and Clem, 1981d), rainbow trout (Elcombe et al., 1985), European perch (Perca fluviatilis L.) (Whittington, 1993), flounder (Paralichthys olivaceus) (Bang et al., 1996), and Southern bluefin tuna (Thunnus maccoyii Castelnau) (Watts et al., 2001). Some of the LMW Ig (160–180 kDa) contains H chain with slightly smaller size (50–75 kDa) than those (60–80 kDa) of the HMW Ig (Clem and McLean, 1975; Elcombe et al., 1985; Watts et al., 2001), while some of the serum LMW Ig (120–140 kDa) has a significantly shorter H chain (40–45 kDa) (Clem, 1971; Lobb and Clem, 1981d). As shown inTable 2, in the case of the sheepshead (Lobb and Clem, 1981a, 1981d), the serum HMW Ig (∼700 kDa) contains two subpopulations, one is a disulfide linked (covalent) form, and the other one is assembled with two noncovalent subunits of disulfide linked dimers (∼350 kDa), while the LMW Ig (∼140 kDa) is composed of two subunits of halfmers (H1L1) (∼70 kDa) associated to one another non-covalently. Notably, the H chain (∼45 kDa) of the LMW Ig could not be recognised by the polyclonal antibody (pAb) developed against the HMW Ig. In early studies, the half-lives of both HMW and LMW Ig of teleost serum were found to be around 12–16 days (Avtalion et al., 1973; Lobb and Clem, 1981a). In a recent study, the high-affinity, highly polymerized trout IgM was shown to have longer halflives than the lower-affinity, lightly polymerized IgM (Ye et al., 2010). Up until recently, it has been widely accepted that the HMW Ig in the serum of teleost is tetrameric IgM. However, the molecular nature of the LMW Ig has been a mystery since its identification 40 years ago. One could suggest, as other authors did in previous studies (Clem, 1971; Clem and McLean, 1975), that the LMW Ig is the in vivo precursor/redox form or the in vitro degradation product of the HMW Ig. Alternatively, the monomeric LMW Ig may represent an unknown Ig isotype different from IgM, based on the fact that the H chain of LMW Ig was, in some cases, structurally and/or antigenically distinct from that of HMW Ig. For example, although the HMW and LMW Ig forms of rainbow trout had similar binding affinities for a hapten, significant differences were revealed in the size, peptide maps, immunoreactivities of their H chains with the pAb against their corresponding H chains. More importantly, the abilities to activate complement between the HMW and LMW Ig forms differed markedly (Elcombe et al., 1985). Furthermore, in sheepshead the HMW and LMW Igs appeared not to be metabolic products of one another, which suggested that they are the products of separate genes (Lobb and Clem, 1981a). However, it was difficult at that time to clarify the relationship between the HMW and LMW Igs and to identify the exact Ig isotype of the LMW Ig due to limited genetic information available for Ig isotypes in teleost. One of the two Ig isotypes (IgD and IgT/IgZ) discovered later in teleost might solve the mystery on the identification of the LMW Ig. Since 1997, the H chain gene (ighı) of the second Ig isotype (IgD) has been identified in all studied teleost, which has shown variable genomic structure and splicing pattern among species (Hordvik, 2002; Saha et al., 2004; Srisapoome et al., 2004; Wilson et al., 1997). Surprisingly, the gene encoding a secretory molecule was only found in the genome of channel catfish (Bengten et al., 2002), and interestingly, in catfish serum the secreted molecule (∼105, 150 or 180 kDa) lacked the V region and the C1 domain, instead it contained only the Fc portion (C1234567sec or C1(234)2567sec) (Bengten et al., 2002; Edholm et al., 2010a). Furthermore, there was no evidence that the catfish secretory molecules associated with L chains to form a typical Ig containing equimolar H and L chains (see the review by Edholm et al., 2011). Thus it would seem unlikely that the LMW Ig is a secretory IgD, however, since the gene organisation of IgD in teleosts is very variable, the possibility that the LMW Ig corresponds to an IgD in some species cannot be completely overruled. Excitingly, the gene (igh/igh) encoding the H chain of a third teleost Ig isotype (IgT/IgZ) was discovered in the genomes of rainbow trout (Hansen et al., 2005), zebrafish (Danio rerio) (Danilova et al., 2005), fugu (Takifugu rubripes) (Savan et al., 2005b), and carp (Savan et al., 2005a) in 2005. After that, similar genes have been identified in almost all studied species belonging to the main orders of teleost fish (reviewed in Zhang et al., 2011) except catfish (Bengten et al., 2006); the completion of the catfish genome may finally reveal whether or not IgT is present in this species. So far, the physicochemical features and functional roles of IgT/IgZ have not been widely investigated except for the case of trout IgT (Zhang et al., 2010). Trout serum IgT is a monomer with a molecular mass similar to those of the above mentioned LMW Ig described in the same species (Elcombe et al., 1985) as well as in other teleosts (Clem and McLean, 1975; Watts et al., 2001). Thus, if the serum LMW Ig is actually a monomeric IgT/IgZ, its H chains (70–75 kDa) in margate (Clem and McLean, 1975) and tuna (Watts et al., 2001) may be predicted to have four C domains like that of rainbow trout (Hansen et al., 2005) and most other teleost, while the H chains (∼45 kDa) of putative serum IgT/IgZ in giant grouper (Clem, 1971) and sheepshead (Lobb and Clem, 1981d) would presumably contain two C domains like that of fugu IgT (Savan et al., 2005b) or carp chimeric IgM–IgT (Savan et al., 2005a). It is clear that to demonstrate the identity of the LMW Ig in teleost, more detailed molecular and biochemical analyses are required. For example, the genomic information of each igh locus needs to be completed at least in the most important model species; the mono-specific Abs against each Ig isotype (IgM, IgT/IgZ, and IgD) should be developed; and the physicochemical features and functional roles of each Ig isotype need to be investigated globally. Moreover, we anticipate that new conclusive data could be obtained by re-analysing previous LMW Ig studies. Besides H chain heterogeneities in teleost serum Ig, several L chain isotypes/variants have also been identified. At the protein level, IgL masses are available from Southern bluefin tuna (∼28, 29 kDa) (Watts et al., 2001), rainbow trout (∼24, 26 kDa) (Sanchez and Dominguez, 1991), Atlantic salmon (Salmo salar) (∼25, 27 kDa) (Havarstein et al., 1988), European perch (∼27–30 kDa) (Whittington, 1993), and channel catfish (F, ∼22/24 kDa; G, ∼26 kDa; , ∼27 kDa; , unknown size) (Edholm et al., 2010a, 2009; Lobb et al., 1984). Such differences were identified based both on molecular size and structural/antigenic analyses. Interestingly, up to four L chain variants (∼27–30 kDa) were found in perch HMW Ig preparations, while only the lightest two L chains (∼27, 28 kDa) were found in the LMW Ig population (Whittington, 1993). This bias raises the questions of whether certain L chain isotypes preferentially associate to a particular H chain isotype (for example, , , or in trout) and whether that happens in specific tissues or at different developmental stages in response to different types of pathogens. In teleosts, Ig genes (F and G in catfish) are the most abundantly expressed in PBLs, with only 2% of IgM+ cells expressing Ig and Ig (Edholm et al., 2009). However, with the exception of the catfish (Edholm et al., 2010a, 2010b, 2009; Lobb et al., 1984) very little is known with regards to which particular L chain isotypes correspond the protein bands detected by SDSPAGE, and thus, specific antibodies against these light chains are required to solve this issue. For further details on the molecular and genomic characterization of teleost L chains please refer to (Hikima et al., 2010; Pilstrom, 2002) (see also the review by Edholm et al., 2011).
3.2. Immunoglobulins in intestine and bile
Until now Ig present in the gut mucus of teleosts has been poorly studied, in part due to methodological challenges imposed by the large amounts of proteolytic enzymes in the collected gut mucus, as reported in Atlantic salmon (Hatten et al., 2001). Nonetheless, differences between gut mucus/bile and serum Ig have long been acknowledged. In the bile of sheepshead, a dimeric Ig (∼320 kDa) was described with H chains of 55 kDa, which appeared to be different from either the serum tetrameric Ig or the skin mucus dimeric Ig, based on differences in its antigenicity and H chain size (Lobb and Clem, 1981b). In rainbow trout, when analysed by gel filtration, the IgM is tetrameric in both serum and gut mucus, while the IgT in serum and gut mucus is chiefly monomeric and polymeric respectively. Interestingly, by SDS-PAGE under nonreducing conditions, the polymeric gut mucus IgT migrates as a monomer, thus indicating that the monomeric subunits of gut polymeric IgT are associated by non-covalent interactions (Zhang et al., 2010). This situation in gut polymeric IgT differs from that of IgM in which its monomers are for the most part associated by covalent (disulfide) bonds (Kaattari et al., 1998). Moreover, while the concentration of IgM in serum (2.5 mg ml−1) was found to be much higher than that in gut mucus (0.075 mg ml−1), the concentration of IgT in gut mucus (0.007 mg ml−1) was double to that of serum. This suggested that teleost IgT could have an important role in gut mucosal immunity as demonstrated in the same study (Zhang et al., 2010). Interestingly, in sheepshead, the concentration (0.09 mg ml−1) of total bile Ig (HMW plus LMW Ig) detected with a polyclonal antibody (Lobb and Clem, 1981a) was comparable with that (0.082 mg ml−1) of trout gut mucus Ig (IgM plus IgT) (Zhang et al., 2010). Further details on the biochemical structure of gut mucus IgT and IgM are provided in Rombout et al. (2010) and Zhang et al. (2010, 2011).
3.3. Immunoglobulins in skin and gill
Compared with the high concentration of serum Ig in teleost, Ig is present at a very low concentration (8–90 g ml−1) in the skin mucus (Hatten et al., 2001; Lobb and Clem, 1981a; Rombout et al., 1986). In terms of spatial distribution, Ig levels in channel catfish were found to be highest on lateral skin, between the gill cover to the dorsal fin, lower between pectoral to anal fins, and lowest on the tail and ventral skin (Zilberg and Klesius, 1997). In the skin mucus of sheepshead, two Igs of different molecular weights were observed (Lobb and Clem, 1981c), one was tetrameric (∼700 kDa), similar to the serum HMW Ig, and the other one was dimeric. A portion (∼350 kDa) of the dimeric Ig consisted of monomeric units associated by non-covalent bonds, while the other portion (400–500 kDa) was covalently linked and associated with a protein (∼95 kDa). This protein was presumed to be a secretory component (SC) of the polymeric Ig receptor (pIgR), but its size is in disagreement with that of the SC recently identified in fugu (∼60 kDa) (Hamuro et al., 2007) and trout (∼35 kDa) (Zhang et al., 2010). The ratio of LMW to HMW Ig was 3 times higher in the skin mucus than serum in sheepshead (Lobb and Clem, 1981a). In olive flounder (P. olivaceus), a monomeric Ig was detected in the cutaneous mucus with a monoclonal antibody (mAb) against a L chain of serum Ig purified with mannan-binding protein (MBP) affinity column (Palaksha et al., 2008; Shin et al., 2007). In carp, the Ig from cutaneous mucus had different protein/carbohydrate composition and antigenicity from that of serum IgM, and the electophoretic analysis revealed that the majority of both Igs were tetrameric (Rombout et al., 1993b). One could speculate that this tetrameric cutaneous Ig in carp may correspond to one of the two recently identified carp IgZ (IgZ1 and IgZ2 (Ryo et al., 2010; Savan et al., 2005a)). In rainbow trout skin mucus, besides the homogeneous redox forms of tetrameric IgM (∼800 kDa) found in serum, a unique redox form consisting of halfmeric constituents (H1L1, ∼100 kDa) has been reported (Bromage et al., 2006). Studies in catfish, on the other hand, did not reveal apparent differences between skinmucus and serum Igs, that is, denaturation of the tetrameric IgM produced eight redox forms, the smallest being a halfmer and the largest a fully linked tetramer (Lobb, 1987). Thus far biochemical analyses on gill mucus immunoglobulins are lacking, although specific IgM responses have been described in the gill mucus from a very small number of teleost species ((Lumsden et al., 1993, 1995) and see Section 5.3 of this review). Thus, a detailed biochemical characterization of gill immunoglobulins is required to have a better understanding of mucosal immune responses in these areas. In that regard, it will be interesting to ascertain whether IgT/IgZ is the prevalent immunoglobulin in gill and skin mucus, as it has been found to be the case in the gut mucus of rainbow trout (Zhang et al., 2010).
3.4. Synthesis and transport of immunoglobulins in mucosal sites
Once structural differences between mucosal and systemic Igs in teleost were found, the question “where do mucosal antibodies come from?” arose. Early studies in sheepshead addressed the metabolic relationships of the Igs found in the serum, skin mucus, and bile by monitoring intravenously injected radiolabeled serum Ig. The authors concluded that the Igs in skin mucus and bile were not derived from the HMW or LMW Ig in serum through simple transudation or active transport, and therefore the Ig in mucosal secretions must have been the result of local synthesis (Lobb and Clem, 1981a). In other teleost species, Ig found in liver extracts suggested that sIg could be transported across the hepatocytes, to be secreted into the bile (Abelli et al., 2005; Jenkins et al., 1994; Rombout et al., 1986). In that regard, it is well known that mammalian sIg can reach the luminal area of the gut through the bile, which contains sIg taken up from plasma by liver hepatocytes that use pIgR for the transcytosis process (Brown and Kloppel, 1989). In carp, skin mucus IgM specific mAbs were reactive with bile capillaries and ducts but not with the intestinal epithelium, thus suggesting that hepatobiliary transport might be the main route used by IgM found in the intestine of this species (Rombout et al., 1993b). In the same species, the above mAbs were immunoreactive with the skin epithelium and the H chain of mucus Ig but not or hardly with the H chain of serum Ig, indicating differences in the composition of the H chains of both molecules and therefore the IgM in skin mucus must have been produced locally (Rombout et al., 1993b). However, in naïve rainbow trout, a number of both IgM+ and IgT+ B cells could be observed in the LP and epithelium of the gut, an observation that suggested a role of some of these cells in producing Ig locally (Zhang et al., 2010). Local production of immunoglobulins in the GALT probably occurs in other species as a number of studies have found B cells in the GALT of fish (see below in Section 4.2), In mammals, sIgs (secretory pentameric IgM or dimeric IgA) are synthesized and secreted by the plasma cells localised at either the MALT or the liver, and then transcytosed through the epithelial layer into the gut lumen or other mucosal sites by the pIgR expressed on the surface of epithelial cells. After transport of the sIg-pIgR complex into the apical pole of the cells, the complex is cleaved off and released into the luminal area. At that point, the sIg remains covalently bound to a portion of the pIgR designated as the SC (reviewed in Brandtzaeg, 2009; Brandtzaeg et al., 2008; Rojas and Apodaca, 2002). The pIgR seems to play a pivotal role in the secretory system of fish. Thus far, pIgR orthologues have been identified in fugu (Hamuro et al., 2007), common carp (Rombout et al., 2008), orange-spotted grouper (Epinephelus coioides) (Feng et al., 2009), and rainbow trout (Zhang et al., 2010). Functional studies have shown that teleost pIgR binds to IgM (Feng et al., 2009; Hamuro et al., 2007; Zhang et al., 2010), as well as to IgT (Zhang et al., 2010). In rainbow trout, gut mucus IgM and IgT were shown to be associated to the SC of the pIgR, whereas serum Ig was devoid of this association, thus implying a role of trout pIgR for the transport of sIg into the gut luminal area (Zhang et al., 2010). These data resembled that of the report on fugu pIgR, in which skin mucus IgM was found associated to a fugu pIgR fragment (Hamuro et al., 2007). The identification of pIgR orthologues in common carp (Rombout et al., 2008) and orange-spotted grouper (E. coioides) (Feng et al., 2009) further indicated that the mucosal Igs of teleost, like mammalian polymeric Igs, need to be transported by a pIgR, although the teleost pIgR only consists of two Ig-like domains, which correspond to the first and fifth domains of mammalian pIgR, respectively (for more extensive reviews on fish pIgR see Rombout et al., 2010; Zhang et al., 2011).