A novel IgA-like immunoglobulin in the reptile Eublepharis macularius

The appearance of antibody genes over evolution coincided with the origin of the vertebrates. Reptiles are of great interest in evolution since they are the link between the amphibians, birds, and mammals. This work describes the presence of a gene in the reptile leopard gecko (Eublepharis macularius) where phylogenetic studies suggest that it is the gene orthologue of immunoglobulin A (IgA) and immunoglobulin X (IgX) in Xenopus. Messenger RNA samples taken from different tissues showed expression of this antibody in intestinal tissue. Data on the structure deduced from the sequence of nucleotides showed an antibody with four domains in the constant region. There is a sequence of 20 amino acids in the C terminus similar to the secretory tail of immunoglobulin M (IgM) and IgA. A detailed analysis of the sequence of amino acids displayed a paradox, i.e., domains CH1 and CH2 showed a clear homology with domains CH1 and CH2 of immunoglobulin Y (IgY) while domains CH3 and CH4 were homologous with domains CH3 and CH4 of IgM. This homology pattern is also seen in Xenopus IgX and bird IgA. The most logical explanation for this phenomenon is that a recombination between the IgM and IgY gave rise to the IgA.     

Molecular characterization of immunoglobulin D in mammals: immunoglobulin heavy constant delta genes in dogs, chimpanzees and four old world monkey species

Antibodies are adaptor molecules that neutralize pathogens and link humoral and cellular defence mechanisms. Immunoglobulin D (IgD), one of the five antibody classes present in mammals, is expressed as an antigen receptor on naïve B cells. The functional role that IgD plays in the immune response is still poorly understood, but the recent characterization of immunoglobulin heavy constant delta genes (IGHD) in a variety of species challenges the view that IgD is of minor importance and is not present in many animals. On the basis of serological studies, IgD appears to be expressed in the majority of mammalian species examined. To confirm, at the molecular level, that IgD is present in different species, we cloned and sequenced IGHD cDNA from dogs and five non‐human primate species (chimpanzee, rhesus macaque, cynomolgus macaque, baboon and sooty mangabey). Our results show that in all six species, IgD heavy chains possess three immunoglobulin domains and a long hinge region encoded by two exons. Only the hinge region of non‐human primates is similar to the human hinge region, with conservation of O‐glycosylation sites and multiple charged residues at opposing ends. The preservation of IgD in primates, dogs and previously characterized species suggests an important functional role for IgD, possibly involving binding to a receptor. The high degree of similarity existing between the structural features of human and non‐human primate IgD suggests that non‐human primates are suitable for in vivo studies designed to define the role that IgD plays in the immune response.     

The impact of ancestral tetraploidy on antibody heterogeneity in salmonid fishes

Summary: The immunoglobulin heavy chain locus in teleost fish is structurally similar to that in mammals, comprising a series of variable gene segments upstream of two constant region genes coding for IgM and IgD. Atlantic salmon have been shown to possess two distinct heavy chain loci, related to the tetraploid ancestry of this fish family. The nature (and results) of the evolutionary processes following the tetraploidization event are the focus of this review. Salmonid fish did not return quickly to a diploid state, but are still in the process of re-establishing disomic inheritance. Thus, a specific locus in one species may still be endowed with four alleles, while it may have been converted to a pair of isoloci in another species. Analyses of immunoglobulin heavy chain genes in Atlantic salmon (Salmo salar) have strongly indicated that the ancestral heavy chain locus was subjected to tetrasomy throughout the radiation of the genera Oncorhynchus and Salmo, and that disomic inheritance was established in the Salmo lineage in the comparatively recent past. The introduction of disomic inheritance at these loci has resulted in two subsets of IgM and IgD heavy chains in Atlantic salmon.     

Zebrafish immunoglobulin IgD: unusual exon usage and quantitative expression profiles with IgM and IgZ/T heavy chain isotypes

The zebrafish is an emerging model for comparative immunology and biomedical research. In contrast to the five heavy chain isotype system of mice and human (IgD, IgM, IgA, IgG, IgE), zebrafish harbor gene segments for IgD, IgM, and novel heavy chain isotype called IgZ/T which appears restricted to bony fishes. The purpose of this study was to design and validate a suite of quantitative real time RT-PCR protocols to measure IgH expression in a vertebrate model which has considerable promise for modeling both pathogenic infection and chronic conditions leading to immune dysfunction. Specific primers were designed and following verification of their specificty, relative expression levels of IgD, IgM, and IgZ/T were measured in triplicate for zebrafish raised under standard laboratory conditions. During embryonic stages, low levels of each heavy chain isotype (IgH) were detected with each increasing steadily between 2 and 17 weeks post fertilization. Overall IgM>IgZ>IgD throughout zebrafish development with the copy number of IgM being several fold higher than that of IgD or IgZ/T. IgD exon usage was also characterized, as its extremely long size and presence of a stop codon in the second IgD exon in zebrafish, raised questions as to how this antibody might be expressed. Zebrafish IgD was found to be a chimeric immunoglobulin, with the third IgD exon spliced to the first IgM constant exon thereby circumventing the first and second IgD exons. Collectively, the qRT-PCR results represent the first comparative profile of IgD, IgM, IgZ/T expression over the lifespan of any fish species and the primers and assay parameters reported should prove useful in enabling researchers to rapidly quantify changes in IgH expression in zebrafish models of disease where altered IgH expression is manifested.     

IgD+IgM- B cells mount immune responses that exhibit altered antibody repertoire

IgM and IgD expression during B cell development and differentiation is strictly and developmentally controlled. Although studies have suggested subtle differences in B cell activation, tolerance, and affinity maturation when antigens ligate cell membrane IgM or IgD, the mechanisms that may explain these differences remain unknown and no drastic differences in immune responses have been reported in mice whose B cells selectively lack IgM or IgD. We now show that the antibody repertoire in IgM(-/-) mice is dramatically altered during the primary response to phosphorylcholine.In IgM(-/-) mice, B cells that are activated and differentiate into antibody-forming cells and germinal center B cells express VH genes other than the T15 genes that dominate in wild-type mice. The kinetics of the antigen-specific IgD primary antibody response in IgM(-/-) mice appears similar to that of IgG, but not to that of IgM in wild-type mice. Thus, our studies demonstrate that differences in the roles played by IgM and IgD in regulating the responsiveness and differentiation of B lymphocytes can have major biological consequences during adaptive immune responses.     

Insights into the function of IgD

IgD, previously thought to be a recent addition to the immunoglobulin classes, has long been considered 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 immunity. However, during the past decade extensive sequencing of vertebrate genomes has shown that IgD homologs are present in all vertebrate taxa, except for birds. Moreover, recent functional studies indicate that IgD likely performs a unique role in vertebrate immune responses. The goal of this review is to summarize the IgD gene organization and structural data, which demonstrate that IgD has an ancient origin, and discuss the findings in catfish and humans that provide insight into the possible function of this elusive immunoglobulin isotype.     

Insights into the function of IgD

IgD, previously thought to be a recent addition to the immunoglobulin classes, has long been considered 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 immunity. However, during the past decade extensive sequencing of vertebrate genomes has shown that IgD homologs are present in all vertebrate taxa, except for birds. Moreover, recent functional studies indicate that IgD likely performs a unique role in vertebrate immune responses. The goal of this review is to summarize the IgD gene organization and structural data, which demonstrate that IgD has an ancient origin, and discuss the findings in catfish and humans that provide insight into the possible function of this elusive immunoglobulin isotype.     

Lymphocyte surface IgD and IgM in Macaca monkeys: ontogeny, tissue distribution and occurrence on individual lymphocytes.

A proportion of lymphocytes in blood, spleen and lymph nodes of nonhuman primates had immunoglobulin on their surfaces detectable by fluorescent antibody to human IgM and IgD. The majority of the individual lymphocytes having either IgM or IgD on their surfaces possessed both classes of immunoglobulin. Lymphocyte surface IgD was capped independently of surface IgM on the same cell when incubated at 37 degrees with anti-IgD. Lymphocytes with surface IgM and/or IgD were present in blood at birth and the percentages over the first 6 months of life were increased compared to older monkeys. A corona of cells faintly positive for both IgM and IgD was observed around germinal centres of both lymph nodes and spleen.     

Expressed IgH μ and τ transcripts share diversity segment in ranched Thunnus orientalis

It is now appreciated that in addition to the immunoglobulin (Ig)M and D isotypes fish also make the mucosal IgT. In this study we sequenced the full length of Ig τ as well as μ in the commercially important Thunnus orientalis (Pacific bluefin tuna), the first molecular analysis of these two Ig isotypes in a member of the order Perciformes. Tuna IgM and IgT are each composed of four constant (CH) domains. We cloned and sequenced 48 different variable (VH) domain gene rearrangements of tuna immunoglobulins and grouped the VH gene sequences to four VH gene segment families based on 70% nucleotide identity. Three VH gene families were used by both IgM and IgT but one group was only found to be used by IgM. Most interestingly, both μ and τ clones appear to use the same diversity (DH) segment, unlike what has been described in other species, although they have dedicated IgT and IgM joining (JH) gene segments. We complemented this repertoire study with phylogenetic and tissue expression analysis. In addition to supporting the development of humoral vaccines in this important aquaculture species, these data suggest that the DH–JH recombination rather than the VH–DH recombination may be instructive for IgT versus IgM/D bearing lymphocyte lineages in some fish.     

Zebrafish immunoglobulin IgD: Unusual exon usage and quantitative expression profiles with IgM and IgZ/T heavy chain isotypes

The zebrafish is an emerging model for comparative immunology and biomedical research. In contrast to the five heavy chain isotype system of mice and human (IgD, IgM, IgA, IgG, IgE), zebrafish harbor gene segments for IgD, IgM, and novel heavy chain isotype called IgZ/T which appears restricted to bony fishes. The purpose of this study was to design and validate a suite of quantitative real time RT-PCR protocols to measure IgH expression in a vertebrate model which has considerable promise for modeling both pathogenic infection and chronic conditions leading to immune dysfunction. Specific primers were designed and following verification of their specificty, relative expression levels of IgD, IgM, and IgZ/T were measured in triplicate for zebrafish raised under standard laboratory conditions. During embryonic stages, low levels of each heavy chain isotype (IgH) were detected with each increasing steadily between 2 and 17 weeks post fertilization. Overall IgM > IgZ > IgD throughout zebrafish development with the copy number of IgM being several fold higher than that of IgD or IgZ/T. IgD exon usage was also characterized, as its extremely long size and presence of a stop codon in the second IgD exon in zebrafish, raised questions as to how this antibody might be expressed. Zebrafish IgD was found to be a chimeric immunoglobulin, with the third IgD exon spliced to the first IgM constant exon thereby circumventing the first and second IgD exons. Collectively, the qRT-PCR results represent the first comparative profile of IgD, IgM, IgZ/T expression over the lifespan of any fish species and the primers and assay parameters reported should prove useful in enabling researchers to rapidly quantify changes in IgH expression in zebrafish models of disease where altered IgH expression is manifested.     

Mouse immunoglobulin receptors on lymphocytes: identification of IgM and IgD molecules by tryptic cleavage and a postulated role for cell surface IgD.

The two mouse immunoglobulin receptors on lymphocytes (IgM and IgD-like) were individually digested by trypsin. The tryptic susceptibility, and the products released, were similar to those of their human counterparts. Evidence for a structural homology between human IgD and its presumed mouse conterpart has been provided by the ramarkably similar profile of fragments resulting from digestion. More definitive homology awaits sequence determination. The extreme susceptibility of surface IgD to proteolysis contrasted with the resistance of surface IgM. We therefore propose that the major role of IgD is to release a fragment (Fabdelata) following exposure to antigen and then elicit a regulatory anti-idiotype response which acts through recognition of the protease-resistant IgM idiotype remaining on the cell surface.     

Analysis and characterization of the expression of the secretory and membrane forms of IgM heavy chains in the pufferfish, Takifugu rubripes

We investigated the structure and expression of immunoglobulin genes in the pufferfish, Takifugu rubripes, a highly prized and economically important fish species. The cDNA fragment that partially encodes the constant region of the IgM heavy chain was isolated in these animals by RACE using degenerate primers after which it was used as a probe for screening IgM heavy chains in a fugu splenic cDNA library. The structural feature of the constant region of fugu sIgM was found to consist of four constant domains (CH1 to CH4), while mIgM was shown to contain a deletion of the CH4 domain, and its transmembrane domain was directly spliced to the CH3 domain as found in other teleosts. This feature may be common to all teleosts. In addition, five VH genes isolated in this study fell into two families based on their variability. Analysis of genomic sequences from the fugu genomic database also showed that there are only two VH families in the genome. The IgM gene was preferentially expressed in presumptive lymphoid tissues. Moreover, in situ hybridization revealed that large numbers of IgM positive cells were widely distributed throughout the spleen, head kidney, kidney, and thymus, confirming that these tissues were major sites of antibody production in fish. The expressions of IgM in the mucosal organs such as the skin, gills, and intestine suggest that they, too, contribute to humoral immunity in aquatic animals. The expression of IgM mRNA in the early development stages of this fish suggests that its larval form possesses a protective defense mechanism against foreign invaders.     

In Vivo Study of mIgM and mIgD Cross-Linking on Murine B Cells

In this study, we have analysed in mice the effects on the immune response of in vivo treatment with different rat monoclonal antibodies (MoAb) against IgM and IgD. Although the effects of IgD cross-linking have been studied already, no attempt has been made to characterize the effects of in vivo IgM crosslinking, probably because of the higher IgM serum levels compared to IgD.
We have used a panel of nine monoclonal rat anti-mouse IgM and three anti-IgD antibodies and we have characterized their isotypes, avidities, immunoglobulin (Ig) cross-linking and internalization abilities. Our results show that injection of mice with some rat MoAb against IgM led to an important decrease of IgM serum level and internalization of membrane IgM (mIgM) on almost all B cells. Similarly, treatment with a high-avidity anti-IgD antibody induced disappearance of mIgD on B cells. Treatment with rat MoAb against IgM or IgD led to a synthesis of specific antibodies and there was a direct relationship between the Ig internalization abilities of rat MoAb and the induction of specific antibody production. Finally, treatment with a high-avidity rat MoAb against IgD induced a polyclonal IgE and IgGl secretion. The significance of these results on mIg receptor functions is discussed.