Developmental and Comparative Immunology 35 (2011) 1309–1316
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Developmental and Comparative Immunology
journal homepage: www.elsevier.com/locate/dci
Insights into the function of IgD
Eva-Stina Edholm, Eva Bengten, Melanie Wilson ∗
University of Mississippi Medical Center, 2500 North State Street, Jackson, MS 39216, United States article info abstract
Article history: IgD, previously thought to be a recent addition to the immunoglobulin classes, has long been consid- Available online 15 March 2011 ered an enigmatic molecule. For example, it was debated if IgD had a specific function other than as an antigen receptor co-expressed with IgM on naive B cells and if it had an important role in mammalian Keywords: immunity. However, during the past decade extensive sequencing of vertebrate genomes has shown that Teleosts IgD homologs are present in all vertebrate taxa, except for birds. Moreover, recent functional studies Immunoglobulin indicate that IgD likely performs a unique role in vertebrate immune responses. The goal of this review is IgD to summarize the IgD gene organization and structural data, which demonstrate that IgD has an ancient Alternative splicing B cells origin, and discuss the findings in catfish and humans that provide insight into the possible function of this elusive immunoglobulin isotype. © 2011 Elsevier Ltd. All rights reserved.
1. Introduction (Gambon-Deza et al., 2010), and Atlantic salmon, Salmo salar (Tadiso et al., 2010). However, PCR and genomic sequencing stud- It is well known that teleost B cells share many similarities with ies have failed to isolate a catfish IgT gene homolog. Also, since IgT mammalian B cells, including immunoglobulin (Ig) gene rearrange- transcripts were not found among the ∼500,000 catfish expressed ments, allelic exclusion, production of membrane Ig and secreted sequence tags in the NCBI database it is likely that the issue of Ig forms (reviewed in Bengten et al., 2000, 2006a; Flajnik and catfish IgT will only be resolved once the sequencing and anno- Kasahara, 2010; Miller et al., 1994; Solem and Stenvik, 2006; Warr, tation of the catfish IgH locus (IGH) is completed. Currently, the 1995) and the use of B cell receptor (BCR) accessory proteins CD79a only immunoglobulins described in catfish are IgM and IgD and and CD79b for Ig signal transduction (Edholm et al., 2010; Sahoo catfish IgM, like teleost IgM in general, has been well studied and et al., 2008). Also, it is established that IgM is the predominant shown to be a structural and functional homolog of mammalian Ig isotype found in teleost serum, while the other Ig isotypes, for IgM (Bengten et al., 2000, 2006b; Warr, 1995). In contrast, much example, IgD in channel catfish, Ictalurus punctatus (Bengten et al., less is known about teleost IgD function. Hence, this review will 2002; Edholm et al., 2010) and IgT in rainbow trout, Oncorhynchus focus on IgD and since IgD has often been described as the most mykiss (Zhang et al., 2010), are found in lesser amounts. In most enigmatic vertebrate Ig, the structure and function of teleost IgD is teleost, serum IgM is expressed as a tetramer, although IgM discussed in the context of what we know concerning mammalian monomers have been described in giant grouper, Epinephelus itaira (and other vertebrate) IgD structure and function. (Clem, 1971) and sheepshead, Archosargus probatocephalus (Lobb IgD was first discovered in human serum as a myeloma pro- and Clem, 1981). In contrast, serum IgT is expressed as a monomer tein in 1965 and then in a companion study was shown to be in rainbow trout serum, and a tetramer in gut mucous (Zhang present in normal serum (Rowe and Fahey, 1965a,b). Later it was et al., 2010). At present serum IgD has only been described in identified on the surface of B cells (Van Boxel et al., 1972). Yet a catfish by using Western blot analyses and whether it exists as a unique function, in addition to initiating BCR signal transduction monomer has yet to be determined. Currently, IgT has been iden- in mature naive B cells, was not identified until recently (Chen tified at the cDNA/DNA level in zebrafish Danio rerio (IgZ; Danilova et al., 2009), and the significance or function of IgD has been a et al., 2005), common carp, Cyprinus carpio (Savan et al., 2005a), subject of debate (reviewed in Geisberger et al., 2006). For exam- rainbow trout (Hansen et al., 2005), Japanese pufferfish, Takifugu ple, it was hypothesized that since anti-Ig treatment of mouse rubripes (Savan et al., 2005b), grass carp, Ctenopharyngodon idella immature B cells resulted in clonal anergy or clonal deletion, the (Xiao et al., 2010), three-spined stickleback, Gasterosteus aculeatus signaling through IgD in mature B cells would result in a quali- tative different signal from that of IgM (Nossal et al., 1979; Pike et al., 1982). Support for this notion came from studies which ∗ ␦ Corresponding author. Tel.: +1 601 984 1719; fax: +1 601 984 1708. used anti-Ig or anti-Ig antibodies to crosslink IgM and IgD on E-mail address: [email protected] (M. Wilson). the surface of murine or human leukemia cell lines and these
0145-305X/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.dci.2011.03.002 1310 E.-S. Edholm et al. / Developmental and Comparative Immunology 35 (2011) 1309–1316 experiments demonstrated that anti-IgM treatment resulted in in cow, Bos taurus; sheep, Ovis aries; and pig, Sus scrofa, range from growth inhibition as indicated by decreased DNA synthesis, while 95.4% to 98.7% when compared to their respective C1 exon of each anti-IgD antibodies did not affect proliferation (Ales-Martinez species and in the pig the first CH exon in an IgD transcript can either et al., 1988; Mongini et al., 1989). However, in the 1990s, stud- beaC␦1orC1(Zhao and Hammarstrom, 2003; Zhao et al., 2002). ies using transgenic (knockout) mice that were germline deficient Also, IgD has been identified in other mammalian species such as, for either IgD or IgM demonstrated that IgM or IgD, each by itself dogs (Rogers et al., 2006), horses (Wagner, 2006) and giant pandas was capable of functioning as a BCR and inducing normal B cell (Zhao et al., 2007). As for the non-mammalian taxa, IgD genes and immune responses (Lutz et al., 1998; Nitschke et al., 1993; Roes and cDNAs have been described in the leopard gecko (Eublepharis mac- Rajewsky, 1993). Briefly, transgenic mice lacking IgM, expressed ularius; Gambon-Deza and Espinel, 2008), green anole lizard (Anolis only IgD on pre- and immature B cells, which not only matured carolinensis; Wei et al., 2009), Chinese soft-shelled turtle (Pelodis- normally, but also populated peripheral tissues in a manner similar cus sinensis; Xu et al., 2009), Xenopus tropicalis (Ohta and Flajnik, to that observed in normal control mice that expressed both IgM 2006) and most recently in the platypus (Ornithorhynchus anatinus; and IgD. These IgM knockout mice also mounted a fairly normal Zhao et al., 2009). Notably, phylogenetic analyses demonstrate that antibody response even though antibody affinity maturation was shark IgW is an ortholog of IgD, which further illustrates the ancient delayed. Similar results were also found in the studies using IgD origin of this isotype (Ohta and Flajnik, 2006). IgW was originally knockout mice. Thus, it was concluded that while IgD appears not identified as IgNARC in the nurse shark (Ginglymostoma cirratum; to be required for a normal B cell response, clearly IgD could sub- Greenberg et al., 1996), IgW in the sandbar shark (Carcharhinus stitute for IgM in regards to both the B cell maturation process and plumbeus; Berstein et al., 1996), and IgX in the clearnose skate (Raja immune function, at least in mice. More recently, studies in human eglanteria; Anderson et al., 1999), reviewed by Flajnik and Rumfelt and catfish have provided evidence indicating that IgD, in addition (2000). However, it appears, at least from the present available data, to functioning as an Ag-binding receptor, is involved in immune that cartilaginous fish belonging to the subclass of holocephali may responses to certain pathogens and plays a role as a mediator of lack an IgD ortholog, since our searches of the existing database for innate immunity (Chen et al., 2009). elephant shark (Callorhinchus milii) and a previous study of spotted ratfish (Hydrolagus colliei; Rast et al., 1998) immunoglobulins failed to identify any IgD gene or cDNA sequences. In addition, IgD is not 2. IgD is found in most major taxa of jawed vertebrates and present in the galliforms (chicken, Gallus gallus; Zhao et al., 2000), displays remarkable structural plasticity between species anseriforms (duck, Anas platyrhynchos; Lundqvist et al., 2001) and in some mammals such as the rabbit and opossum (Oryctolagus Before the identification of an IgD homolog in catfish, IgD was cuniculus; Lanning et al., 2003; Monodelphis domestica; Wang et al., believed to represent a recently evolved Ig exclusive to primates 2009; reviewed in Sun et al., 2010). and rodents (Wilson et al., 1997). In fact it was only recently appre- Here, it is also important to emphasize that structural variations ciated that IgD is also found in a wide variety of other vertebrates, of IgD occur between different vertebrates. In placental mammals, including both mammals and ectotherms. Thus, it appears that IgD the Ig␦ genes and the encoded IgD proteins are structurally highly and its orthologs represent an ancient Ig class, most likely as old as conserved (Rogers et al., 2006; White et al., 1985; Zhao et al., 2007). IgM. The catfish IgD homolog was originally identified as a cDNA, For example, all mammalian IgD genes currently sequenced are which consisted of a rearranged VH region (VDJ) spliced to a C1 located directly 3 of the Ig gene and with the exception of rodents domain, followed by seven novel C␦ domains, a transmembrane all consists of eight exons. Exon 1 encodes the first Ig domain (C␦1) (TM) region, and a short cytoplasmic tail (CYT) consisting of five and exons 2 (H1) and 3 (H2) encode the extended hinge region that amino acids, K-V-K-I-A (Wilson et al., 1997). The finding of such is characteristic of most mammalian IgD proteins. The second and a long IgD-like transcript was surprising since human and mouse third Ig domains (C␦2 and C␦3) are encoded by exons 4 and 5; exon IgD have three and two C␦ domains, respectively. A second catfish 6 encodes the hydrophilic secreted tail (␦Sec) and exons 7 and 8 IgD cDNA was also identified in the same cDNA library screen. This encode the membrane tail (␦TM1 and ␦TM2). Mouse and rat IgD cDNA was a partial transcript and it lacked the TM region and ended genes consist of six exons and encode for shorter IgD forms since in a predicted secreted tail (Bengten et al., 2002; Wilson et al., 1997). these species lack the exons encoding H2 and C␦2. Also, while two Overall, the features supporting the classification of these novel hinge regions are present in the pig and giant panda IgD genes, cDNAs as IgD included: (1) sequence similarities to mammalian Ig␦ these species only utilize their first hinge exon (H1) resulting in a as demonstrated by phylogenetic analysis, (2) location of the catfish less extended IgD hinge in these species (supplemental Fig. 1; Zhao Ig␦ gene immediately 3 of the Ig gene in the IGH chain locus, (3) et al., 2003, 2007). co-expression with Ig in the catfish clonal 3B11 B cell line and (4) In contrast, while the IgD genes of ectothermic vertebrates vary the apparent expression of Ig␦ from a long primary transcript con- in their number of C␦ exons, C␦ exon duplications, and the number taining a rearranged VDJ and Ig and Ig␦ genes (Wilson et al., 1997). of IgD gene copies, the size of the expressed IgD in these species is This presence of the C1 domain in all catfish membrane Ig␦ tran- relatively large. The number of unique C␦ domains in these differ- scripts indicated that catfish IgD, like mammalian IgD, is produced ent species ranges from 6 to 11, and some species have two exons by alternative splicing of mRNA and not by mechanisms involving that encode their TM and others have only a single TM exon. Also, classical isotype switching by chromosomal recombination. More- some teleost express IgD transcripts that have blocks of repeated over, the finding that Ig exons of different isotypes (IgM and IgD), C␦ domains and all teleost IgD molecules examined are chimeric can combine was novel and later it was observed to occur in all since they include C1. None of the cold-blooded vertebrate IgD teleost IgD cDNAs sequenced to date (Fig. 1; Gambon-Deza et al., genes sequenced to date have individual exons that would encode 2010; Hirono et al., 2003; Hordvik, 2002; Hordvik et al., 1999; Saha a hinge, i.e., the C regions are composed of Ig domains. Perhaps, the et al., 2004; Stenvik and Jorgensen, 2000). This inclusion of C1in lack of a hinge in the IgD in these species is compensated by the catfish Ig␦ transcripts was hypothesized to be necessary since it length of the IgD H chain that may allow for some flexibility. would permit the covalent association of Ig␦ H chains with catfish Briefly and beginning with the teleosts, in the catfish the IGH IgL chains, i.e., the C1 domain provides the appropriately located locus spans ∼1 Mbp and contains multiple internal duplications and cysteine required for IgH–IgL chain association. The inclusion of transpositions. It contains three IgD genes termed IGHD1, IGHD2 C1oraC1-like sequence in Ig␦ transcripts is also observed in and IGHD3 and each is individually linked to either a functional artiodactyls. For example, the nucleotide identities of the C␦1 exon Ig gene or an Ig pseudogene (Bengten et al., 2006a,b, 2002). E.-S. Edholm et al. / Developmental and Comparative Immunology 35 (2011) 1309–1316 1311
Fig. 1. Schematic representations of different vertebrate IgD molecules. Comparison of IgD molecules in different species. The C␦ domains are dark grey, black indicates inclusion of C1, dark hatched grey indicates inclusion of C1-like domain, light grey indicates IgL C domains, and white boxes indicate V domains. Hinge regions are indicated by angles and are hatched; the predicted inter-chain disulphide bonds are also shown.
IGHD1 is located directly 3 of the functional C gene and consists C␦3-C␦4 duplication(s) found in ictalurids and salmonids. Instead of 14 exons, with the exons encoding C␦2-C␦3-C␦4 repeated three the pufferfish gene has a longer tandem duplication of C␦1-C␦2- times in tandem: C␦1-(C␦2-C␦3-C␦4)3-C␦5-C␦6-C␦7-TM. This gene C␦3-C␦4-C␦5-C␦6 and the resulting cDNA transcript consisted of encodes the chimeric membrane form of IgD and notably mem- VDJ-C1-(C␦1-C␦2-C␦3-C␦4-C␦5-C␦6)2-C␦7-TM (Fig. 2; Saha et al., brane IgD transcripts do not contain exon duplications. Similar IgD 2004). In the stickleback the IGH locus contains three arrays of VHn- gene organizations with varying numbers of C␦2-C␦3-C␦4 block D-JH-C-D3-J4-C-C␦ in tandem (Bao et al., 2010). The three IgD duplications also occur in Atlantic salmon, Atlantic halibut, grass genes do not contain any exon duplications and are ∼90% identi- carp, fugu and zebrafish (Hordvik, 2002; Hordvik et al., 1999; Saha cal at the amino acid level to each other. However, the C␦5 exon et al., 2004; Xiao et al., 2010). The other two catfish IgD genes in IGHD1 contains a nucleotide deletion. Thus, in stickleback, while are found ∼500 kbp 5 of IGHD1 and are linked with their cor- there are three sets of IGH genes, there is no evidence of a functional responding pseudo C genes. The IGHD2 gene is truncated and difference between the IgD genes. In the salmonids, as a conse- consists of intact C␦1 and C␦2 exons and a C␦3 exon that con- quence of the tetraploid ancestry, there are two IGH loci on separate tains a two-nucleotide deletion creating a frame shift. Currently, chromosomes. For example, in the Atlantic salmon each of the two it is unknown if this gene is functional since IGHD2 transcripts IGH loci, have multiple duplications of the IgT gene, but only one set have not been found, however there is a polyadenylation site 3 of the IgM and IgD genes (Yasuike et al., 2010). The corresponding of the C␦3. IGHD3, is almost identical to IGHD1 except its termi- C␦ exons of the two IgD genes in the IGHA and IGHB loci are very nal exon encodes the secreted IgD tail (␦Sec). Thus, in catfish, one similar to each other and an alignment of C␦ translated exons from gene encodes the IgD membrane form and a second gene encodes Ig␦A and Ig␦1B gives an identity index of 97% (Hordvik, 2002). The secreted IgD and both are associated with functional enhancers. main difference is in the number of C␦2-C␦3-C␦4 tandem duplica- In comparison, different types of IgD gene duplications occur in tions. In Ig␦A there are three C␦2-C␦3-C␦4 tandem duplications and pufferfish, stickleback, Atlantic salmon and Atlantic cod, Gadus in Ig␦B there are four (Yasuike et al., 2010). To date, the Atlantic cod morhua. As in the catfish, the pufferfish membrane and secreted IgD gene appears to be the one exception to the general teleost IgD forms of IgD are encoded by separate genes; the gene encoding structural pattern. It is much shorter and lacks C␦3, C␦4, C␦5, and the putative secreted Ig form is linked to a separate array of D C␦6 exons. Instead the Atlantic cod gene contains a tandem C␦1- and JH genes found 5 of the D and JH gene array linked to the C␦2 duplication separated by an intron containing a short exon Ig and membrane Ig␦ genes (Aparicio et al., 2002). Also, later in termed ␦y. The second C␦1-C␦2 is followed by C␦7 and two TM a second study, which focused on membrane IgD, it was shown exons and full-length transcripts consist of VDJ-C1-C␦1-C␦2-␦y- that the pufferfish membrane IgD gene lacks the tandem C␦2- C␦1-C␦2-C␦7-TM1-TM2 (Stenvik and Jorgensen, 2000). Here, it is 1312 E.-S. Edholm et al. / Developmental and Comparative Immunology 35 (2011) 1309–1316
Fig. 2. Comparison of IgD gene organizations in various teleost. Schematics show only the genomic organization of the C and C␦ genes. Boxes indicate the C and C␦ exons, which are labeled and color-coded. The color-coding of the different exons is based on their phylogenetic relatedness as demonstrated in supplemental Fig. 2. The tandem exon duplications within individual IgD genes are outlined. Drawing is not to scale. important to emphasize that when individual C␦ domains from For teleosts, these transcripts arise by two different mechanisms: different teleost species are compared, it is clear that respective (1) the existence of a separate gene encoding a secreted IgD form domains are homologous, i.e., D1 domains cluster together, D2 as in the catfish and pufferfish or (2) alternative splicing of a mem- domains cluster together, etc. (supplemental Fig. 2). Also, previous brane IgD gene as in the Atlantic salmon. As discussed above and phylogenetic analyses of different teleost C␦ domains suggested in Section 3, catfish secreted IgD is encoded by the IGHD3 gene that exons C␦5-C␦6-C␦7 arose from a gene duplication of exons and pufferfish have a secreted IgD gene upstream of the IgM gene. C␦2-C␦3-C␦4(Gambon-Deza et al., 2010; Hordvik et al., 1999) and Importantly, IgD can be detected in catfish serum by using the teleost C␦1, C␦5 and C␦6 domains clearly are related to human C␦1, anti-IgDsec 2E5 monoclonal Ab (Bengten et al., 2002). As deter- C␦2 and C␦3 domains, respectively (Hordvik et al., 1999; Wilson mined by western blot analyses two forms of secreted IgD of ∼130 et al., 1997). and ∼180 kDa in size is routinely observed in sera from individual IgD in amphibians and reptiles is similar in length to most of catfish and the difference in size may be due to either the inclu- the teleost IgD orthologs. Xenopus IgD transcripts consist of a rear- sion or exclusion of the duplicated C␦2-C␦3-C␦4 domains (Fig. 3; ranged VDJ, eight C␦ domains and a TM; the Xenopus Ig␦ gene Edholm et al., 2010). Based on densitometry of Coomassie blue- contains eight C␦ exons, followed by two TM exons (Ohta and stained gels the concentration of secreted IgD in catfish serum is Flajnik, 2006; Zhao et al., 2006). As for the reptiles, the IgD gene in ∼0.04 mg/ml (Bengten et al., 2002). The evidence for an Atlantic the green anole lizard is single copy and consists of 11 C␦ domains salmon putative secreted IgD form comes from RT-PCR studies, followed by two TM exons (Wei et al., 2009; and supplemental Fig. which identified splice variants where the TM2 exon is spliced 1). Currently, the only transcripts sequenced for the green anole directly to C␦6. This splicing generates a transcript ending in a were obtained by nested PCR and they consists of VDJ-C␦1-C␦2- 14 amino acid tail (Hordvik, 2002). For reptiles, only the leopard C␦3-C␦4-TM1-TM2. In contrast, the leopard gecko has two IgD gecko has been shown to have a secreted IgD form, which was genes that are quite different (Gambon-Deza and Espinel, 2008). identified as a cDNA transcript that ends with the C␦11 exon and The first IgD gene is directly 3 of the IgM gene and contains 11 C␦ contains 24 additional nucleotides that are spliced out of the mem- exons and one TM exon and a membrane IgD transcript shows that brane transcripts. This results in an IgD secreted form with an eight the TM1 exon splices into a cryptic splice within the C␦7 domain. amino acid tail (Gambon-Deza and Espinel, 2008). In contrast, dif- The second IgD gene, termed IgD2, is located ∼20 kb 3 of the first ferent secreted IgD (IgW or IgNARC) forms are more abundantly IgD gene. It contains only seven Ig domains and is predicted to be expressed in the elasmobranch species: nurse shark, horned shark, the result of a partial duplication of the first IgD gene since the Heterodontus francisci, clearnose skate, bounded houndshark, Tri- first four and seventh exons resemble C␦ exons and exons 3 and akis scyllium (Anderson et al., 1999; Honda et al., 2010; Rumfelt 4 resemble IgA-like exons. Transcripts originating from this gene et al., 2004). Likewise, short and long forms of IgD are found in have not been published. the sarcopterygiian dipnoi, as represented by the African lungfish, Interestingly, all cold-blooded vertebrates, except for elasmo- Protopterus annectens (Ota et al., 2003). The IgD genes in the elasmo- branchs, lack a typical mammalian IgD secreted exon that is usually branchs contain six C␦ exons and alternative splicing produces both found 5 of the TM exons (Fig. 2 and supplemental Fig. 1) and tran- long and short forms of secreted and membrane IgD. Specifically, scripts encoding a secreted IgD form are quite rare in these species. long secreted IgD transcripts contain six C␦ exons and short IgD E.-S. Edholm et al. / Developmental and Comparative Immunology 35 (2011) 1309–1316 1313
Fig. 3. Catfish secreted IgD varies in size. Serum from eight different outbred catfish was visualized by Western blot analysis using anti-IgDsec mAb 2E5 followed by goat anti-mouse Ig (H + L) HRP. The different size variants of ∼130 and 180 kDa may be due to the inclusion or exclusion of a repeated block of C␦2, C␦3, and C␦4 exons found within the IGH3 locus that encodes the secreted IgD form. A schematic illustrating the different secreted IgD proteins is shown at left; C␦ domains are in grey and the secreted tail is white. Molecular weight size markers in kDa are shown at right of serum panel. transcripts have only the first two C␦ exons. The short membrane population in humans, i.e., the cells use a cryptic switch region IgD transcripts also only contain the first two C␦ exons, while the found between the C and C␦ genes (Chen et al., 2009). Human long membrane IgD transcripts contain the first four C␦ domains. IgD-only B cells while rare in peripheral blood (∼3.0%), have been Furthermore, long IgD/IgW forms were identified in the nurse shark shown to make up a substantial population (20–25%) of the human by using immunoprecipitation protocols (Greenberg et al., 1996). upper respiratory tract mucosal B cells and in the past 12 years, In the African lungfish membrane IgD forms have yet to be iden- several laboratories have been focusing on this unique IgD-only B tified, but sequencing showed that secreted IgD transcripts either cell population (Chen et al., 2009; Kluin et al., 1995; Owens et al., contain seven C␦ exons or two C␦ exons. Finally, recent annotation 1991; White et al., 1990; Yasui et al., 1989). Most IgD-only B cells of the monotreme duck-billed platypus genome demonstrated that are found associated with tonsillar tissues. The IgD-only B cells are the platypus IgD gene is located between the IgM and IgG1 genes larger, have more cytoplasm than conventional naive IgM+/IgD+ B (Gambon-Deza et al., 2009; Zhao et al., 2009). It consists of 10 C␦ cells, and exhibit a centroblast morphology (Billian et al., 1996; exons followed by two TM exons and to date a secreted exon has Liu et al., 1996). Notably, >90% of these IgD-only B cells utilize not been found. Besides resembling the IgD expressed by teleosts, IgL chains (Arpin et al., 1997) and sequence analyses showed amphibians and leopard gecko in its length, phylogenetic analy- that these cells express highly mutated VH and VL regions. Also, ses and amino acid comparisons show that all of the platypus C␦ since the mutations are found scattered throughout the CDR and domains have a corresponding domain in reptile IgD. Also a domain FR regions it was proposed that the different V regions would prob- by domain comparison demonstrated that platypus C␦1, C␦6 and ably be unable to bind Ag (Liu et al., 1996; Seifert et al., 2009; C␦7 domains are homologous to the mammals C␦1, C␦2 and C␦3 Wilson et al., 1998; Zheng et al., 2005). Moreover, studies per- (Zhao et al., 2009). formed by Chen et al. (2009) demonstrated that secreted IgD made Taken together the available IgD sequencing data indicate, in by the IgD-only B cells is bound to the surface of basophils (and marked contrast to IgM, that IgD genes have undergone numerous mast cells) by a calcium-mobilizing receptor. First, immunofluores- alterations involving both exon duplications and deletions lead- cence staining showed that IgD is found on the surface and in the ing to different IgD structural forms in different species (Hordvik cytoplasm of mucosal and circulating basophils, which indicates et al., 1999; Ohta and Flajnik, 2006; Rogers et al., 2006; Stenvik and IgD-bound immune complexes are internalized. Second, cross- Jorgensen, 2000; Tucker et al., 1980; Wagner et al., 2004; White linking of basophil-bound IgD using an anti-IgD mAb triggered an et al., 1985; Wilson et al., 1997; Zhao and Hammarstrom, 2003; intracellular calcium flux and induced secretion of B cell activation Zhao et al., 2002). factors and cytokines, including BAFF (B cell-activating factor of TNF family), APRIL (a proliferation-inducing TNF ligand), IL-3, and IL-4. 3. IgD-expressing B cells in humans and catfish Third, IgD cross-linking induced basophils to secrete the antimicro- bial factors cathelicidin, pentraxin-3, and -defensin. In addition, Most of what we know about IgD expression and function the authors showed that supernatants collected from IgD cross- comes from studies performed using human and mouse tissues linked basophils inhibited the growth of the respiratory pathogens and cell lines, and knockout mice. Also, it was only after the initial Moraxella catarrhalis and Haemophilus infuenzae. However, even sequencing of IgD in catfish and in other teleosts, followed by the though the putative IgD-binding receptor (Fc␦R) was not isolated, widespread sequencing of different genomes, that IgD was found the authors’ studies suggest that it is a high affinity receptor since in vertebrates other than primates and rodents. However, despite adding exogenous labeled IgD to cultured basophils did not lead the many publications demonstrating that IgD has a more ancient to an increase in surface IgD staining, indicating saturation of the origin than originally thought (see above), very few reports have binding sites. Exogenous IgD could only be bound by the basophils focused on the function and cellular expression of this isotype in after they were stripped of their IgD and IgD binding by pre- vertebrates, except for the studies performed in humans and mice. treated basophils was abolished if the “added” IgD was denatured Consequently, this section summarizes our recent characterization or if stripped basophils were treated with trypsin. Interestingly, studies of catfish IgD-bearing cell populations and how these relate in this same study, patients with autoinflammatory syndromes to human IgD-bearing cells. were shown to have more circulating and mucosal IgD-only B cells In humans (and mice), IgM and IgD are expressed on the surface and more mucosal “IgD-armed” basophils than healthy subjects, of naive mature B cells by alternative splicing of a long primary which suggests that inflammation promotes the switch to IgD. RNA transcript. These double positive B cells (IgM+/IgD+) make Also, cross-linking of IgD-armed basophils (from these patients) up the majority of the peripheral B cells and upon Ag binding, resulted in the increased secretion of both IL-1 and TNF, which sug- they down-regulate their IgD expression. In contrast, the major- gests that IgD bound to the surface of basophils in the respiratory ity of long-lived memory B cells have class switched, undergone mucosa is able to trigger an innate immune response (Chen et al., somatic hypermutation and express either high affinity IgG, IgE, 2009). or IgA (McHeyer-Williams, 2003). Interestingly, class switching is As for catfish IgD, in the same study it was shown by using also involved in generating a unique IgM−/IgD+ (IgD-only) B cell flow cytometry that catfish express two types of IgD+ B cell 1314 E.-S. Edholm et al. / Developmental and Comparative Immunology 35 (2011) 1309–1316
Fig. 4. Catfish express three types of IgD+-bearing B cells. The single IgM+ (IgM+/IgD−), double positive IgM+/IgD+, and IgD+-only (IgM−/IgD+) B cells are depicted with their membrane immunoglobulins associated with CD79 accessory molecules. IgM+ B cells produce secreted IgM tetramers and the IgD+-only B-cells are the main producers of “V-less” secreted IgD (ref). Immunoglobulin domains are represented as in Fig. 2:C, black; C␦, dark grey, CL, light grey; V, white. The CD79a and CD79b Ig domains are stripped and Y marks the location of the two tyrosine residues in the immunoreceptor tyrosine-based activation motifs. populations (see below) and a population of circulating granular restricted to ectothermic vertebrates. Also, even though the cat- cells that are armed with exogenous IgD via a putative IgD-binding fish IgM+/IgD− and IgM+/IgD+ B cell populations have the potential receptor. Currently, the origin of these granular cells is unknown, to utilize any of the four different IgL chain isotypes (IgL F, IgL G, however at the message level these cells did not express message IgL and IgL ) most IgM+ B cells utilize the IgL orthologs, F for either membrane or secreted IgD, IgM, or TCR and they did and G, i.e., only 2–5% of IgM+ B cells isolated from peripheral blood not react with an anti-catfish neutrophil mAb. However, these IgD- express IgL or IgL chains, isotypes known to have very limited granulocytes could not be isolated via direct selection techniques as repertoires in catfish (Edholm et al., 2009). Currently, while the dif- this invariably resulted in degranulation and granulocyte destruc- ferent roles of catfish IgM+/IgD+ and IgD-only B cells have not been tion. Recently, a more detailed characterization of catfish IgD-only defined, we hypothesize that catfish membrane IgD functions as B cells was published by Edholm et al. (2010). Basically, catfish a typical Ag binding receptor. Both cell types express message for express three different types of B cells: single positive IgM+/IgD− B membrane IgD with functionally rearranged VDJ regions and the cells, double positive IgM+/IgD+ B cells and IgD-only B cells (Fig. 4). B cell accessory signaling molecules CD79a and CD79b. Also, co- The IgM+/IgD− and IgM+/IgD+ B cells are small agranular lympho- immunoprecipitation experiments demonstrate that membrane cytes and are morphologically indistinguishable from each other, IgD associates with IgL chains, and requires association of CD79 while the IgD-only B cells resemble human IgD-only B cells. They molecules for B cell surface expression. Furthermore, no bias in the are large, exhibit a plasmablast morphology and have a higher cyto- V, D or J family usage of membrane IgD transcripts was observed in plasm to nucleus ratio as compared to catfish IgM+ B cells and in the different IgD-expressing B cell types. In addition, anti-IgD cross- contrast to mammalian IgD-only B cells, catfish IgD-only B cells linking in IgM+/IgD+ B cells induces a calcium flux, albeit it is slower can represent as much as 60–80% of the total peripheral blood B than the response induced by IgM cross-linking of IgM+/IgD− and cells, depending on the individual fish. Notably, as shown by cell IgM+/IgD+ B cells (Edholm unpublished). Whether this is due to sorting experiments, co-immunoprecipitations, and RT-PCR, cat- lower levels of IgD expression on the surface of IgD+ B cells and/or fish IgD-only B cells exclusively utilize IgL chains, an IgL isotype the nature of the anti-IgD mAb has not been determined. E.-S. Edholm et al. / Developmental and Comparative Immunology 35 (2011) 1309–1316 1315
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