A new isotype of immunoglobulin heavy chain in the urodele amphibian Pleurodeles waltl predominantly expressed in larvae

Up to now, it was thought that urodele amphibians possessed only two IgH isotypes, IgM (mu) and IgY (upsilon). By screening a Pleurodeles waltl Ig cDNA mini-library, we identified three isotypes: IgM, IgY and a previously unknown class. IgM are multimeric molecules and represent the most abundant isotype throughout the life of P. waltl. IgY are likely the counterpart of mammalian IgA. The new isotype has typical Ig H-chain characteristics and is expressed as both secretory and membrane forms. Our analyses indicate that this isotype is restricted to Pleurodeles. Consequently, we named it "IgP" (pi) for Pleurodeles. This isotype is mainly expressed after hatching. Its expression decreases after metamorphosis. Our data indicate that IgP-expressing B cells present some similarities with mammalian B1-cells.     

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.     

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.     

Partial characterization of different cell types found in the Xenopus laevis lymphoreticular tumor based on the presence or absence of surface immunoglobulins and Fc molecules

Cells of Xenopus laevis lymphoreticular tumor induced by tumor tissue transplantation were examined for surface Ig and Fc receptor molecules in order to evaluate the different cell types found in the tumor. Direct immunofluorescent technique, using fluorochrome conjugated rabbit antisera to Xenopus Ig's, detected Ig molecules on the surface of a mean of 31.7 +/- 11.3% of cells in tumor suspensions. Most of these molecules were of IgM isotype, reversibly bound to the cell membrane (cytophilic) and could be dissociated by acid pH or overnight cell culturing. In addition integral membrane IgM was detected on the surface of 10.2 +/- 5.9% of the cells. The serum origin of cytophilic Ig's and the cellular origin of integral membrane Ig's were confirmed by analysis of electrophoretic mobility of their heavy chains on SDS-polyacrylamide gels. The existence of Fc receptor molecules on the surface of 48.6 +/- 16.6% of the cells was demonstrated by fluorescent staining using heat aggregated FITC labelled IgM or FITC or TRITC labelled antigen-complexed IgY antibodies. 32.2 +/- 12.4% and 16.4 +/- 6.8% of the cells bore receptors for IgY or receptors for IgM respectively, while 6.3 +/- 3.1% carried receptors for both Ig's. Double fluorescent staining revealed that 28.9 +/- 4.5% of cells bearing IgM on their surface expressed also receptors for IgY. These results attest to the heterogeneity of the tumor cell population, in respect to the presence or absence of FcR-IgY, FcR-IgM, sIgM, and cytophilic IgM surface molecules.     

Immunoglobulin classes in the garden lizard,

Two classes of immunoglobulins have been purified from lizard serum using a combination of ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200 or on Sepharose 6B. Lizard IgM is 2-ME sensitive and has an intact molecular weight similar to human IgM. On SDS-PAGE, reduced IgM dissociates into heavy and light chains of molecular weight 70,000 and 23,500 daltons respectively. Lizards also possess a 2-ME resistant, low molecular weight immunoglobulin designated as IgY similar to avian and amphibian IgY. IgY dissociates on SDS-PAGE to yield 59,500 dalton heavy and 26,000 dalton light chains. Antisera raised in rabbits to each of the two Ig classes could be made class-specific by cross-absorption, thus indicating that IgM and IgY represent distinct isotypes.     

Immunoglobulin classes in the garden lizard,Calotesversicolor

Two classes of immunoglobulins have been purified from lizard serum using a combination of ion-exchange chromatography on DEAE-cellulose and gel filtration on Sephadex G-200 or on Sepharose 6B. Lizard IgM is 2-ME sensitive and has an intact molecular weight similar to human IgM. On SDS-PAGE, reduced IgM dissociates into heavy and light chains of molecular weight 70,000 and 23,500 daltons respectively. Lizards also possess a 2-ME resistant, low molecular weight immunoglobulin designated as IgY similar to avian and amphibian IgY. IgY dissociates on SDS-PAGE to yield 59,500 dalton heavy and 26,000 dalton light chains. Antisera raised in rabbits to each of the two Ig classes could be made class-specific by cross-absorption, thus indicating that IgM and IgY represent distinct isotypes.     

Autoantibodies to the Nuclear Sp100 Protein in Primary Biliary Cirrhosis and Associated Diseases: Epitope Specificity and Immunoglobulin Class Distribution

Sp100, a protein with a dot-like intranuclear localization in immunofluorescence microscopy, is a major target for patient autoantibodies in primary biliary cirrhosis (PBC) and occasionally in rheumatic disorders. The human Sp100 cDNA has recently been cloned, and the deduced amino acid sequence was found to contain sequence similarities with an MHC class I domain and several transacting regulatory proteins, including HIV-1 nef proteins. In this study, recombinant Sp100 fusion proteins were used to differentiate the immunoglobulin isotypes and to map the epitopes involved in the anti-Sp100 autoimmune response. PBC patients developed IgG as well as IgM and/or IgA class anti-Sp100 autoantibodies whereas most patients with rheumatic diseases developed IgG class autoantibodies only. For epitope mapping, truncated versions of the Sp100 protein were probed for immunoreactivity in ELISA and immunoblotting. With 55 sera, 17 different reaction patterns were obtained, and at least three non-overlapping major autoantigenic domains were recognized by the majority of sera. One domain, which contains the sequence similarity with HIV nef proteins, was recognized by all anti-Sp100 sera and harbours multiple, in part discontinuous, epitopes. These data demonstrate a heterogeneous and patient-specific anti-Sp100 autoimmune response which is antigen-driven and, at least in terms of isotype composition, different in PBC and non-PBC patients.     

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.     

Bile immunoglobulin of the duck (Anas platyrhynchos). II. Antibody response in influenza A virus infections

The capacity of the IgM-like bile immunoglobulin (IgX) of the duck (Anas platyrhynchos) to express antibody activity to H3N2 influenza A viruses, and the dependence of this activity on the co-existence of serum IgM antibodies were investigated. Ducklings infected orally and intranasally at 15-29 days of age with viruses isolated from different host species were examined for haemagglutination-inhibiting (HI) antibodies in biles and sera 16-29 days after infection (p.i.). All biles had antibodies associated with IgX; all sera had antibodies associated only with the 7.8S IgG. Following oral infection of birds 42-days-old with influenza A/duck/HK/7/75 virus, serum HI antibodies were an initial IgM response occurring from 5-12 days p.i., followed by the appearance of 7.8S IgG antibodies. Virus-neutralizing (VN) antibodies in serum were also biphasic; isotype classification was not attempted. Bile IgX developed HI and VN activity. HI antibodies reached peak titres 12 days p.i. and fell to low levels by 24 days p.i. VN antibodies also reached peak titres 12 days p.i., but thereafter persisted at quite high levels throughout the experiment. Development of high titres of antibody in bile coincided with the termination of virus excretion in faeces. These experiments confirm that bile IgX of the duck can function as antibody in response to influenza A viruses, and that its activity appears to be independent of serum IgM. Its possible relevance in determining survival of virus in the intestine is discussed.     

Shark immunity bites back: affinity maturation and memory response in the nurse shark, Ginglymostoma cirratum

The cartilaginous fish are the oldest phylogenetic group in which all of the molecular components of the adaptive immune system have been found. Although early studies clearly showed that sharks could produce an IgM-based response following immunization, evidence for memory, affinity maturation and roles for the other isotypes (notably IgNAR) in this group remained inconclusive. The data presented here illustrate that the nurse shark (Ginglymostoma cirratum) is able to produce not only an IgM response, but we also show for the first time a highly antigen-specific IgNAR response. Additionally, under appropriate conditions, a memory response for both isotypes can be elicited. Analysis of the response shows differential expression of pentameric and monomeric IgM. Pentameric IgM provides the 'first line of defense' through high-avidity, low-affinity interaction with antigen. In contrast, monomeric IgM and IgNAR seem responsible for the specific, antigen-driven response. We propose the presence of distinct lineages of B cells in sharks. As there is no conventional isotype switching, each lineage seems pre-determined to express a single isotype (IgM versus IgNAR). However, our data suggest that there may also be specific lineages for the different forms (pentameric versus monomeric) of the IgM isotype.     

Axolotl MHC architecture and polymorphism.

The MHC of the urodele amphibian Ambystoma mexicanum consists of multiple polymorphic class I loci linked, so far as yet known, to a single class II B locus. This architecture is very different from that of the anuran amphibian Xenopus. The number of class I loci in the axolotl can vary from 6 to 21 according to the haplotypes as shown by cDNA analysis and Southern blot studies in families. These loci can be classified into seven sequence groups with features ranging from the class Ia to the class Ib type. All individuals express genes from at least three of the seven groups, and all individuals possess the class Ia-like type.     

Elevated Levels and Different Repertoire Profile of Colostral Anti-LPS Antibodies May Have a Significant Role in Compensating Newborn Immunity

A high prevalence of systemic infections caused by enterobacteria such as Escherichia coli is observed during the neonatal period. Lipopolysaccharide (LPS) is one of the major factors responsible for septic shock caused by these Gram-negative bacteria. We have recently demonstrated the presence of anti-LPS immunoglobulin (Ig)G antibodies in cord blood with a repertoire identical to that found in maternal serum. In the present study, we analyzed anti-LPS O111 antibody isotypes in maternal serum and colostrum from mothers and in cord serum from their respective full-term (n = 30) and preterm (n = 13) neonate infants. The main isotype found in serum samples from mothers of term infants was IgM (range between 28 and 54 mg/l), followed by IgA (1-2 mg/l) and IgG (2-3 mg/l). The range of IgG antibody concentrations in cord blood was between 2 and 3 mg/l, as a result of placental transfer. A novel observation in our study was that the LPS bands recognized by colostral antibodies were completely different from those recognized by IgG in serum. Colostral IgA antibodies recognized several bands not bound by serum IgG antibodies from the respective maternal serum, independently of the antibody quantity. In addition, we verified the pattern of LPS recognition by serum IgA and colostral IgA antibodies was identical, what suggested that the antibody isotype found in serum could probably be derived from differentiated IgA-positive cells which were homing to the mucosa through the mucosal homing mechanism. Identical pattern of recognition was obtained comparing the IgA and IgM isotypes in colostrum. Slight differences in the pattern of recognition were found between colostral and serum IgM antibodies. The fact that colostral antibodies recognize much more bands than serum antibodies may be important for the host to mount an effective immune response in the intestinal lumen, in order to prevent excessive absorption of LPS, reducing possible systemic effects caused by the molecule.     

Suppression of polyclonal immunoglobulin production by M-proteins shows isotype specificity

Monoclonal gammopathies are B cell neoplasms that are characterized by the presence of monoclonal immunoglobulins (M-proteins) in the serum. By an unknown mechanism, the normal polyclonal immunoglobulin levels are frequently reduced in sera of these patients. To assess the role of M-protein isotype in this effect, we used serum protein electrophoresis to quantitate monoclonal and polyclonal immunoglobulins in patients and we used serum immunofixation electrophoresis to determine their M-protein isotype. When divided into populations of 30 patients with IgG M-proteins (mean 2.5 g/dl) and 19 patients with IgM or IgA M-proteins (mean 2.6 g/dl), the mean polyclonal immunoglobulin level was significantly lower in the IgG M-protein population (0.4 g/dl) than the IgM/IgA population (0.8 g/dl). Patients with IgG M-proteins also had significantly lower polyclonal immunoglobulin levels when compared separately with the patients with either IgA or IgM paraproteins. Since the polyclonal immunoglobulin fraction is comprised mostly of IgG, these results give the first direct indication that IgG M-proteins have a greater suppressive effect on polyclonal IgG levels than do M-proteins of other isotypes. These findings suggest that an isotype-specific feedback mechanism could be involved in the normal regulation of serum IgG levels.     

Allotype-associated differences in concentrations of human IgG subclasses

The concentrations of seven immunoglobulin isotypes (IgA, IgE, IgM, IgG1, IgG2, IgG3, and IgG4) were measured in the sera of 207 Finnish blood donors, and they were allotyped with anti-Gm antibodies: anti-f, anti-a, anti-x, and anti-n. The above population could be divided into 12 phenotypes, and significant differences in isotype concentrations between different phenotypes were observed. They are best explained by postulating that the following alleles of different loci are associated with a high concentration of the product of the locus: a(x)-IgG1, n-IgG2, b-IgG3, and perhaps 4b-IgG4. The following concentration differences between the low and the high homozygotes were found: IgG1, 1.2-fold; IgG2, 1.5-fold; and IgG3, 2.6-fold. No significant allotype-associated differences in the concentrations of IgA, IgM, or IgE could be detected.     

Tryptic digestion of Xenopus IgM and IgY molecules

Xenopus IgM and IgY molecules were digested by trypsin. Their respective fragments were separated by gel filtration and immunoadsorption. The purified fragments were characterized by SDS-PAGE and immunoblotting. Tryptic digestion of Xenopus IgM resulted in the release, at a low yield, of hexameric Fcmu, and of monovalent Fabmu fragments. The digestion of Xenopus IgY antibodies led to the recovery of divalent and monovalent Fab nu fragments. The antigen-binding property of these fragments was demonstrated. No Fc nu fragments of appreciable size could be detected.     

Progression to type 1 diabetes is associated with a change in the immunoglobulin isotype profile of autoantibodies to glutamic acid decarboxylase (GAD65). Childhood Diabetes in Finland Study Group

To investigate whether type 1 diabetes in man is associated with a preferential Th1/Th2 response, and whether autoantibodies to one of the main autoantigens would reflect such a response, we characterized the immunoglobulin isotype profile to the 65-kDa isoform of glutamic acid decarboxylase (GAD65) in siblings to IDDM patients. Samples obtained from affected subjects before and at clinical onset of IDDM, from unaffected individuals at high risk and at low risk and from healthy controls were studied. The immunoglobulin isotype profile in the siblings at low risk reflected a more immature, i.e., IgM and Th2 like, i.e., IgE response compared to the progressors and siblings at high risk, with significantly higher median levels of IgM and IgE. The rank order of anti-GAD65 immunoglobulin isotypes was similar in the siblings before and at clinical onset of IDDM, IgG1 > IgG4 > IgM > IgE > IgA > IgG3 > IgG2, but markedly different in the individuals at low risk, IgG1 > IgM > IgE > IgG4 > IgG3 > IgA > IgG2. Based on these observations, we suggest that progression to clinical onset of IDDM is associated with a maturation and a decrease in the Th2 immune response against GAD65; findings which could have implications for future intervention and prediction strategies.