Immunoglobulins of the non-galliform birds: antibody expression and repertoire in the duck

Galliform and non-galliform birds express three immunoglobulin isotypes, IgM, IgA and IgY. Beyond this we should not generalize because differences in gene organization may have functional consequences reflected in the immune response. At present, studies on non-galliform birds are largely restricted to ducks. Ducks express an alternatively spliced form of their IgY heavy chain (upsilon) gene, the IgY(DeltaFc), that lacks the Fc region and Fc-associated secondary effector functions. It is not known how common the expression of the IgY(DeltaFc) is among birds, nor the functional consequences. It is also not known whether the unusual organization of the duck IgH locus, also shared with the chicken, having the gene order of mu, alpha and upsilon, with alpha inverted in the locus, is unique to the galloanseriform lineage. Ducks, like chickens, have a single immunoglobulin light chain of the lambda (lambda) type. Evidence suggests that ducks, like chickens, generate their immunoglobulin repertoire through a single functional rearrangement of the variable (V) region, and generate diversity through gene conversion from a pool of pseudogenes. In Southern blots of germline and rearranged bursal DNA, both the heavy and light chain loci of ducks appear to each undergo one major rearrangement event. For both heavy and light chains, the functional V region element and the pseudogenes appear to consist of a single gene family. Further analysis of 26 heavy chain joining (JH) and 27 light chain JL segments shows there is use of a single J segment in ducks, which is diversified presumably through somatic mutations and gene conversion events. Despite this limitation on the rearrangement of immunoglobulin genes, analysis of 26 DH and 122 VL sequences suggests that extensive sequence diversity is generated.     

Characterization of IgA and IgM binding and internalization by surface-expressed human Fcα/μ receptor

The Fcα/μ receptor (Fcα/μR) is an unusual Fc receptor in that it binds to two different antibody isotypes, IgA and IgM. This receptor is of interest because it is thought to be involved in the capture of IgA- and IgM-immune complexes and antigen presentation. To further characterize this receptor, we were able to stably express human Fcα/μR on the surface of the 293T cell line. Using this system, we determined the affinity of the interactions of the receptor with IgA and IgM, which led to novel insights including the important finding that IgM polymers can bind to human Fcα/μR in the absence of J chain. This is in contrast to the polymeric immunoglobulin receptor (pIgR), which requires the presence of J chain to bind to polymeric IgA and IgM. The dissociation constants (K_d) of all of the different human IgA isotypes and allotypes for human Fcα/μR were determined, and we show that the N-linked glycans on IgA1 are not required for binding to the receptor. In addition, we demonstrate that IgA can be rapidly internalized by human Fcα/μR in the presence of cross-linking antibody.     

Molecular and functional characteristics of the Fcα/μR, a novel Fc receptor for IgM and IgA

IgM is the first antibody to be produced in a humoral immune response and is a major isotope of natural antibodies and may play an important role in innate immunity. On the other hand, IgA is a secreted antibody at the mucosal membrane such as the gastrointestinal and respiratory tracts and protects from initial invasion of microbes. However, how these antibodies are involved in immunity has been poorly elucidated. We previously identified a novel Fc receptor for IgA and IgM, designated Fcα/μ receptor (Fcα/μR), whose gene is closely located at the polymeric immunoglobulin receptor (poly-IgR), also a receptor for IgA and IgM, in the Fc receptor gene cluster on the chromosome 1. In contrast to the the poly-IgR that is expressed on epithelial, but not hematopoietic, cells, Fcα/μR is constitutively expressed on the majority of B lymphocytes and macrophages in the spleen and at the center of the secondary lymphoid follicles. The Fcα/μR mediates endocytosis Staphylococcus aureus /anti-S. aureus IgM antibody immune complexes by B lymphocytes, for which the dileucine motif in the cytoplasmic tail of the mouse Fcα/μR is responsible. These results reveal a new mechanism in the primary stage of immune defense against microbes.     

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.     

Characterization of IgA and IgM binding and internalization by surface-expressed human Fcα/μ receptor

The Fcα/μ receptor (Fcα/μR) is an unusual Fc receptor in that it binds to two different antibody isotypes, IgA and IgM. This receptor is of interest because it is thought to be involved in the capture of IgA- and IgM-immune complexes and antigen presentation. To further characterize this receptor, we were able to stably express human Fcα/μR on the surface of the 293T cell line. Using this system, we determined the affinity of the interactions of the receptor with IgA and IgM, which led to novel insights including the important finding that IgM polymers can bind to human Fcα/μR in the absence of J chain. This is in contrast to the polymeric immunoglobulin receptor (pIgR), which requires the presence of J chain to bind to polymeric IgA and IgM. The dissociation constants (K(d)) of all of the different human IgA isotypes and allotypes for human Fcα/μR were determined, and we show that the N-linked glycans on IgA1 are not required for binding to the receptor. In addition, we demonstrate that IgA can be rapidly internalized by human Fcα/μR in the presence of cross-linking antibody.     

Differing surface marker characteristics in plasma cell dyscrasias with particular reference to IgM myeloma.

Immunological marker studies using direct immunofluorescence and rosette methods were performed on the bone marrow and peripheral blood of an IgM myeloma, and on the peripheral blood of two cases of IgA myeloma and one IgG myeloma. These studies confirmed the peripheral blood involvement in plasma cell dyscrasias. In the IgA myeloma, the membrane and cytoplasmic immunoglobulin was restricted to IgA, and the IgG myeloma did not express immunoglobulin at the surface of the cells shown to contain cytoplasmic IgG. The IgM myeloma cells, on the other hand, expressed membrane immunoglobulin, including IgD, of a single light chain type. The majority of plasma cells secreting IgA or IgG did not express the IgG Fc receptor, but most of the cells from the IgM myeloma, including plasma cells, did express IgG Fc receptors.     

Expression of heavy chain‐only antibodies can support B‐cell development in light chain knockout chickens

Since the discovery of antibody‐producing B cells in chickens six decades ago, chickens have been a model for B‐cell development in gut‐associated lymphoid tissue species. Here we describe targeting of the immunoglobulin light chain locus by homologous recombination in chicken primordial germ cells (PGCs) and generation of VJC_L knockout chickens. In contrast to immunoglobulin heavy chain knockout chickens, which completely lack mature B cells, homozygous light chain knockout (IgL^?/?) chickens have a small population of B lineage cells that develop in the bursa and migrate to the periphery. This population of B cells expresses the immunoglobulin heavy chain molecule on the cell surface. Soluble heavy‐chain‐only IgM and IgY proteins of reduced molecular weight were detectable in plasma in 4‐week‐old IgL^?/? chickens, and antigen‐specific IgM and IgY heavy chain proteins were produced in response to immunization. Circulating heavy‐chain‐only IgM showed a deletion of the CH1 domain of the constant region enabling the immunoglobulin heavy chain to be secreted in the absence of the light chain. Our data suggest that the heavy chain by itself is enough to support all the important steps in B‐cell development in a gut‐associated lymphoid tissue species.     

Is Xenopus IgX an analog of IgA?

Using class specific monoclonal antibodies we analyzed the tissue distribution of B cells expressing the three immunoglobulin (Ig) isotypes (IgM, IgX, IgY) in Xenopus. Large numbers of IgM- and IgX-, but not IgY-, positive B cells are located in the gut epithelium of the intestine. In this organ up to 60% of all B cells can be IgX positive, while in the spleen or liver they are hardly detectable. The majority of IgX-producing cells resemble plasma cells. IgY-producing cells are found in the liver and spleen but not in the intestine. In contrast to IgY, the expression of IgM and IgX is thymus independent. Upon systemic immunization, a several-fold increase of specific IgM and IgY, but not IgX, antibodies was detected in the sera. This and its association with the mucosae of the intestine resembles results reported for mammalian IgA; therefore, IgX of Xenopus might be considered an analog of IgA.     

IgX antibodies in the urodele amphibian Ambystoma mexicanum

Until recently, it was believed that urodele amphibians are able to synthesize only two immunoglobulin isotypes, IgM and IgY. We reinvestigated this issue in the Iberian ribbed newt Pleurodeles waltl and reported recently that this urodele expresses at least three isotypes: IgM, IgP and IgY. In this study, we demonstrate that another urodele, Ambystoma mexicanum, has also a third isotype whose amino acid sequence presents the highest homology with the amino acid sequence of Xenopus IgX. This isotype has typical Ig H-chain characteristics, could form multimers and is mainly expressed in mucosal tissues thereby indicating that it is likely the physiological counterpart of Xenopus IgX and mammalian IgA. Interestingly, no IgP could be found in A. mexicanum, in contrast to P. waltl, in which IgX was not found in previous investigations. These data indicate, for the first time, that different families of urodeles can express different immunoglobulin isotypes.     

The diagnostic value of serological studies in pediatric patients with acute Mycoplasma pneumoniae infection

BackgroundMycoplasma pneumoniae is a common pathogen of respiratory tract infections in pediatric patients. Serological studies are traditional methods for the diagnosis. However, early diagnosis of M. pneumoniae infections remains problematic. We investigate the value of early serum immunoglobulin A (IgA), in addition to immunoglobulin G (IgG), and immunoglobulin M (IgM) levels, in children infected with M. pneumoniae.MethodsFrom August 2016 to February 2017, we enrolled pediatric patients based on both clinical symptoms and chest x-ray, and confirmed by positive throat culture for M. pneumoniae. Serum titers of M. pneumoniae IgM, IgG, and IgA during the acute phase were checked. All respiratory samples were further analyzed by polymerase chain reaction (PCR). Diagnostic values of different tests were evaluated.ResultsFifty-six patients fulfilled the diagnostic criteria, with a median age of 4.84 years. Most of them (89.3%) were enrolled within 7 days of disease onset. PCR was positive in 71.4% of the study population. Early IgG samples were of limited value in diagnosing M. pneumoniae infection, of which 89.3% showed a negative result. Positive rates of early serum IgA and IgM were 48.2% and 46.4%, respectively. In combination with IgA and/or IgM, the sensitivity increased to 71.4% during their early clinical course.ConclusionsIn the pediatric population, combined serological tests of M. pneumoniae IgA and IgM, offer an accurate method of early diagnosis comparable to that of PCR, and can be an alternative choice for prompt detection of mycoplasma infections when PCR and culture are not available.     

Quantification of human immunoglobulins by semiautomatic polyethylene glycol precipitation radioimmunoassays. Estimation of circulating immune complexes and immunoglobulin synthesized in vitro

Simple, reliable semiautomatic radioimmunoassays have been developed for the measurement of human immunoglobulins (IgG, IgA, IgM, IgK and Ig lambda). The assays are based on the separation of free from antibody-bound radiolabelled fragments of immunoglobulin by precipitation with 13% polyethylene glycol. The precipitate is harvested and washed on glass fibre filters by means of a commercial cell harvester. Radiolabelled lambda chain from Bence Jones urine was used for lambda chain assay and Fab' gamma from pooled IgG for the kappa assay. Labelled Fab or Fc fragments of IgG, IgA and IgM were used for the class specific assays. Selected commercial antisera were used throughout. The sensitivity limits of the assays performed according to the standard procedure were about 20 ng Ig per ml. The assays have been used for the quantification of circulating immune complexes and both cellular and secreted immunoglobulin produced in cell cultures. The results demonstrate the importance of the simultaneous quantification by L chain and H chain specific assays.     

Evidence for an early appearance of modern post-switch isotypes in mammalian evolution; cloning of IgE,IgG and IgA from the marsupial Monodelphis domestica

In birds, reptiles and amphibians the IgY isotype exhibits the functional characteristics of both of IgG and IgE. Hence, the gene for IgY most likely duplicated some time during early mammalian evolution and formed the ancestor of present day IgG and IgE. To address the question of when IgY duplicated and formed two functionally distinct isotypes, and to study when IgG and IgA lost their second constant domains, we have examined the Ig expression in a non-placental mammal, the marsupial Monodelphis domestica (grey short-tailed opossum). Screening of an opossum spleen cDNA library revealed the presence of all three isotypes in marsupials. cDNA clones encoding the entire constant regions of opossum IgE (ϵ chain), IgG (γ chain) and IgA (α chain) were isolated, and their nucleotide sequences were determined. A comparative analysis of the amino acid sequences for IgY, IgA, IgE and IgG from various animal species showed that opossum IgE, IgG and IgA on the phylogenetic tree form branches clearly separated from their eutherian counterparts. However, they still conform to the general structure found in eutherian IgE, IgG and IgA. Our findings indicate that all the major evolutionary changes in the Ig isotype repertoire, and in basic Ig structure that have occurred since the evolutionary separation of mammals from the early reptile lineages, occurred prior to the evolutionary separation of marsupials and placental mammals.     

Defense genes missing from the flight division

Birds have a smaller repertoire of immune genes than mammals. In our efforts to study antiviral responses to influenza in avian hosts, we have noted key genes that appear to be missing. As a result, we speculate that birds have impaired detection of viruses and intracellular pathogens. Birds are missing TLR8, a detector for single-stranded RNA. Chickens also lack RIG-I, the intracellular detector for single-stranded viral RNA. Riplet, an activator for RIG-I, is also missing in chickens. IRF3, the nuclear activator of interferon-beta in the RIG-I pathway is missing in birds. Downstream of interferon (IFN) signaling, some of the antiviral effectors are missing, including ISG15, and ISG54 and ISG56 (IFITs). Birds have only three antibody isotypes and IgD is missing. Ducks, but not chickens, make an unusual truncated IgY antibody that is missing the Fc fragment. Chickens have an expanded family of LILR leukocyte receptor genes, called CHIR genes, with hundreds of members, including several that encode IgY Fc receptors. Intriguingly, LILR homologues appear to be missing in ducks, including these IgY Fc receptors. The truncated IgY in ducks, and the duplicated IgY receptor genes in chickens may both have resulted from selective pressure by a pathogen on IgY FcR interactions. Birds have a minimal MHC, and the TAP transport and presentation of peptides on MHC class I is constrained, limiting function. Perhaps removing some constraint, ducks appear to lack tapasin, a chaperone involved in loading peptides on MHC class I. Finally, the absence of lymphotoxin-alpha and beta may account for the observed lack of lymph nodes in birds. As illustrated by these examples, the picture that emerges is some impairment of immune response to viruses in birds, either a cause or consequence of the host-pathogen arms race and long evolutionary relationship of birds and RNA viruses.     

Molecular and functional characteristics of the Fcalpha/muR, a novel Fc receptor for IgM and IgA

IgM is the first antibody to be produced in a humoral immune response and is a major isotope of natural antibodies and may play an important role in innate immunity. On the other hand, IgA is a secreted antibody at the mucosal membrane such as the gastrointestinal and respiratory tracts and protects from initial invasion of microbes. However, how these antibodies are involved in immunity has been poorly elucidated. We previously identified a novel Fc receptor for IgA and IgM, designated Fcα/μ receptor (Fcα/μR), whose gene is closely located at the polymeric immunoglobulin receptor (poly-IgR), also a receptor for IgA and IgM, in the Fc receptor gene cluster on the chromosome 1. In contrast to the the poly-IgR that is expressed on epithelial, but not hematopoietic, cells, Fcα/μR is constitutively expressed on the majority of B lymphocytes and macrophages in the spleen and at the center of the secondary lymphoid follicles. The Fcα/μR mediates endocytosis Staphylococcus aureus /anti-S. aureus IgM antibody immune complexes by B lymphocytes, for which the dileucine motif in the cytoplasmic tail of the mouse Fcα/μR is responsible. These results reveal a new mechanism in the primary stage of immune defense against microbes.     

Radioimmunochemical studies on 7.8S and 5.7S duck immunoglobulins in comparison with Fab and Fc fragments of chicken IgY

In ducks two forms of low molecular weight Ig's +), 7.8S and 5.7S, have been described. To compare their antigenicity with Fab and Fc fragments of chicken IgY a double-antibody RIA technique was used in which the binding of 125I-chicken IgY or 125I-chicken IgY (Fc) to rabbit and carp anti-chicken IgY (Fab) and anti-chicken IgY (Fc) antibodies, respectively, was inhibited by duck 7.8S and 5.7S Ig's. Chicken IgY and duck 7.8S Ig ( IgY -like protein) are highly cross-reactive with respect to their Fab as well as Fc part determinants. On the other hand, the duck 5.7S Ig shows nearly identical determinants to the Fab fragments of 7.8S Ig, but a Fc part is lacking. Therefore, we conclude that the 5.7S Ig molecule in ducks doesn't represent a separate Ig class. It consists of the same principal type of heavy chains as the 7.8S Ig, but the H chain of the 5.7S Ig lacks very likely the last two homologous constant regions.     

Induction of Fc receptors for IgA on murine T cell hybridoma by human monoclonal IgA and by high molecular weight IgA in IgA nephropathy.

A reproducible immunocyto-adherence assay has been developed to study the modulation of Fc receptors for IgA (Fc alpha R), using a murine T cell hybridoma (T2D4), which expresses Fc receptors for all known isotypes of secreted immunoglobulins. By using sheep red blood cells coated with the hapten 2-4-6 trinitrophenyl (TNP), as indicator cells, and a murine monoclonal IgA (MOPC 315) antibody with anti-TNP activity, we were able to study the Fc alpha R on T2D4 cells. We found that: (a) murine Fc alpha R can bind human monoclonal IgA, and this binding is isotype specific since it was inhibited by human monoclonal IgA but not by human monoclonal IgG or IgM; (b) the expression of murine Fc alpha R is unducible by human monoclonal IgA, and this effect is isotype specific since it is not observed with human monoclonal IgM or IgG (c) sera from patients with IgA nephropathy can also induce Fc alpha R expression; by contrast, no induction was observed with normal human sera, (d) in one serum from an IgA-nephropathy patient, the inducer factor was characterized by affinity chromatography on anti-IgA-Sepharose and by gel filtration: high molecular weight IgA, probably IgA aggregates or immune complexes were recognized to be responsible for the induction of murine Fc alpha R expression.     

Evaluation of Newcastle disease virus immunoassays for waterfowl using a monoclonal antibody specific for the duck immunoglobulin light chain

In the present study a monoclonal antibody (mAb 14A3) was tested for its reactivity against serum immunoglobulin Y (IgY) of several waterfowl species, and subsequently for its applicability as anti-species antibody in common immunoassays. Western blot analyses demonstrated its broad cross-reactivity with the serum IgY light chain of different duck species: Muscovy duck (Cairina moschata), Mallard (Anas platyrhynchos), white-winged wood duck (Asarcornis scutulatus), common pintail (Dafila acuta). Reactivity was also evident with IgY of two swan species—mute swan (Cygnus olor) and black-necked swan (Sthenelides melanocoryphus)—and two goose species—domestic goose (Anser anser var. domestica) and red-breasted goose (Rufibrenta ruficollis). Applying the mAb for Newcastle disease virus (avian paramyxovirus serotype 1 [APMV-1]) test systems, its functionality within indirect immunoassays was evaluated. Using APMV-1-positive sera of domestic geese and Muscovy ducks, mAb 14A3 facilitated specific staining of APMV-1-infected cells in an immunofluorescence test. In addition, it proved to be functional in an indirect enzyme-linked immunosorbent assay (ELISA) and a western blot assay. Thus, the analysed mAb represents an attractive and versatile reagent that offers the opportunity to develop serological tests for waterfowl, allowing a high sample throughput using the ELISA technique or the fine analysis of humoral immune responses using the western blot.     

Receptors for immunoglobulin isotype on T and B lymphocytes from untreated patients with chronic lymphocytic leukaemia

Peripheral blood lymphocytes from eighteen untreated patients with chronic lymphocytic leukaemia (CLL) were analysed for the proportions of T and B lymphocytes with receptors for IgM, IgG or IgA. T lymphocytes with Fc receptors for IgM (T mu cells) or IgA (T alpha) cells were found in proportions comparable to those found in the controls. However, the proportion of T lymphocytes with receptors for IgG (T gamma cells) was significantly increased (P < 0.001) resulting in an abnormally low ratio of T mu/T gamma (P < 0.001), when compared with normal controls. The proportion of B cells bearing Fc receptors for IgM, IgG or IgA was determined simultaneously. No significant differences were found between the normal controls and the patients with CLL. In vitro treatment of the purified T and B lymphocyte preparations with human leucocyte interferon, did not alter the proportions of the lymphocytes expressing Fc receptors for various immunoglobulin isotypes. The significance of these findings is discussed.