Comparative studies on the structure of biliary immunoglobulins of some avian species. II. Antigenic properties of the biliary immunoglobulins of chicken, turkey, duck and goose

Studies on the immunological cross-reactivity of the Igs+ from the bile of chicken and turkey (order Galliformes) as well as duck and goose (order Anseriformes) to Igs with antigenic known properties carried out by a series of RIA. Various class-specific anti-Ig sera produced in rabbits, guinea-pigs and mice were used. The following results were obtained: (1) A high extent of antigenic relationship between the chicken and turkey biliary Igs was demonstrated by a radiobinding system consisting of 125I-turkey biliary Ig and class-specific rabbit anti-turkey biliary Ig antibodies. The biliary Igs of duck and goose did not inhibit this antigen binding system. (2) The duck and goose biliary Igs were able to inhibit the binding of 125I-carp IgM to Fc-fragment-specific rabbit anti-carp IgM antibodies and of 125I-human IgM to Fc-fragment-specific guinea-pig anti-human IgM antibodies, respectively. (3) None of the biliary Igs show cross-reactive properties common with human and porcine IgA. That was detected by inhibition of the radio-binding system from 125I-human IgA and alpha chain-specific mouse anti-human IgA antibodies. Therefore, the conclusion was made that in anseriform birds IgM-like Igs are the secretory Igs, while in galiform birds biliary Igs with special antigenic properties occur.     

Evolution of low molecular weight immunoglobulins--III. The immunoglobulin of chicken bile--not an IgA

Studies on comparison of antigenic relationships between the immunoglobulin of chicken bile--the so-called chicken IgA--and human, porcine, as well as murine IgA were performed by a series of double-antibody and solid-phase RIA systems using H chain-specific anti-chicken biliary Ig sera and alpha chain-specific anti-human IgA sera produced in rabbits and in carp. It was not possible to demonstrate a significant immunological cross-reactivity of the Ig of chicken bile to the Ig's of IgA class. Besides, we could not find an Ig type similar to this chicken biliary Ig in pooled normal sera or in the crude Ig fractions precipitated by (NH4)2SO4 of several vertebrate species (man, swine, buzzard, duck, goose, kestrel, kite, three tortoise species, frog, carp and perch); or in the bile of duck, goose, two snake species and frog. Only pooled chicken normal serum contained this biliary Ig type. Accordingly, the chicken biliary Ig can no longer be considered a precursor of mammalian IgA. This Ig of chicken bile represents a unique Ig class of birds and is to be called IgB (dominant biliary Ig of some birds).     

cDNA sequence and organization of the immunoglobulin light chain gene of the duck, Anas platyrhynchos

A cDNA was cloned which encoded an immunoglobulin (Ig) light (L) chain of the White Pekin duck. The organization of the variable (V) and constant (C) domains was analyzed by genomic Southern blotting. The duck L chain gene has a similar chromosomal organization to that of the chicken, with a single λ-like C region and multiple V_L hybridizing elements. The amino acid sequence of the V_L region of the White Pekin duck L chain showed 88% identity with the Muscovy duck and 87% identity with the chicken, the J_L region showed 92% identity with these species, and the C_L region showed 88% identity with Muscovy duck and 66% with chicken. The constraints imposed by the gene-conversion mechanism of generating antibody diversity might account for the similarities of the avian V region sequences.     

Evolution of low molecular weight immunoglobulins. V. Degree of antigenic relationship between the 7S immunoglobulins of mammals, birds, and lower vertebrates to the turkey IgY

There are close antigenic relations between the turkey IgY--the dominant 7S Ig of serum--and the corresponding IgY-like Igs of chicken, duck, goose, tortoise, and frog as well as serum or colostral IgA of man and swine. The RIA-systems used for the determination of immunological cross-reactivity among the Ig types consisted of 125-I-labelled turkey IgY and class-specific rabbit anti-turkey IgY antibodies. The results stand in a good agreement with our findings demonstrating strong antigenic relationships between various serum or colostral Igs of IgY and IgA types, respectively, to the chicken IgY. Therefore, the IgY-like Igs seem to be the precursors for the IgA class of mammals.     

The efficiency of cysteine-mediated intracellular retention determines the differential fate of secretory IgA and IgM in B and plasma cells

Previous studies on IgM secretion demonstrated a role for the μ chain C-terminal cysteine (Cys575) in preventing the transport of unpolymerized subunits along the secretory pathway. The sequence homology between the C-terminal tailpieces of μ and α heavy chains prompted us to investigate the role of cysteine-mediated retention in the control of IgA secretion during B cell development. Similar to IgM, IgA are not secreted by B lymphocytes: the retention mechanism can be reversed by the reducing agent 2-mercaptoethanol, suggesting that disulfide interchange reactions are involved in the quality control of both IgM and IgA. Yet, α2L2 subunits, but not μ2L2, are secreted constitutively by plasma cells. We demonstrate that the differential retention of IgM and IgA subunits by myeloma transfectants is mainly due to the presence of an acidic residue upstream the α chain C-terminal cysteine. The regulation of polymeric Ig secretion during B cell development provides an example of how thiol-mediated quality control can be modulated according to the aminoacidic context surrounding the critical cysteine and to the cell type.     

Channel catfish soluble FcμR binds conserved linear epitopes present on Cμ3 and Cμ4

A linear epitope on catfish IgM has been identified as the docking site for the catfish soluble FcμR (IpFcRI). Western blot analyses and latex bead binding assays identified the consensus octapeptide motif FxCxVxHE located at the second cysteine that forms the intrachain disulfide bond of the catfish Cμ3 and Cμ4 immunolglobulin (Ig) domains as the IpFcRI binding sites. Furthermore, molecular modeling of catfish Cμ3 and Cμ4 confirmed that the octapeptide in both of these domains is accessible for IpFcRI interactions. In addition, since this octapeptide motif is also found in other vertebrate Ig domains, IpFcRI binding to Ig heavy (H) and light (L) chains from rainbow trout, chicken, mouse, rabbit, and goat were examined by Western blot analyses and latex bead binding assays. IpFcRI readily bound reduced rainbow trout (Igμ), chicken (Igν), mouse (Igμ, Igγ1, Igγ2a, Igγ2b, and Igα), rabbit (Igμ and Igγ) and goat (Igγ) IgH chains, and mouse Igκ and Igλ, and chicken Igλ IgL chains. IpFcRI also bound mouse IgM, IgA and IgG subclasses when examined under native conditions.     

DIVERSITY IN DNA REARRANGEMENTS AND IN RNA EXPRESSIONS OF IMMUNOGLOBULIN GENE ON COMMON VARIABLE IMMUNODEFICIENCY

Six heterogeneous common variable immunodeficiency (CVID) patients were analysed for germ-line DNA, DNA rearrangements, and RNA expressions of immunoglobulin (Ig) gene by Southern or northern blotting using appropriate probes. We detected no polymorphism in neutrophil DNA hybridized to a C μ and a C γ probe. In three patients, both serum Ig and Ig-bearing cells were scarcely detected, and by northern hybridization methods, neither μ mRNA, γ mRNA, α mRNA nor k mRNA was detected. However, one Epstein-Barr virus-transformed B lymphoblastoid cell line (LCL) of these three patients was different from the germ line in the region of J_H, Cγ, and Ck , and expressed μ mRNA at a higher level. The B cell defects of these three patients lay on the B cell maturation stage similar to X-linked agammaglobulinaemia (XLA). In two others among the six CVID patients, serum IgM and IgM-bearing cells were detected to a certain degree, and by northern hybridization, μ mRNA was detected at a lower level, but neither μ mRNA, α mRNA, nor k mRNA was detected. One LCL of these two patients could express μ mRNA at the normal level. In the last patient, the serum IgM was normal, serum IgG and IgA were somewhat low, Ig-bearing cells were normal, μ mRNA and k mRNA were detected at the normal level, and γ mRNA and k mRNA were detected at a lower level. The defect of this patient affected the class switch stage. These results showed that primary B cell defects in CVID occurred at several B cell differentiation stages which could be classified by expression of the Ig gene, and at the degree of clonal diversity in the B cell repertoire. Furthermore, this study provides support for the idea that the CVID defect is related to a more generalized cellular function, such as regulating the proliferation and/or clonal expansion of cells of the B lymphoid lineage.     

Monoclonal IgM, IgA and IgG in the serum of a single individual: immunofluorescence identification of cells producing the immunoglobulins.

Monoclonal IgM(?) and IgA(?) in the serum of patient CM were previously shown to share identical, individually specific (Ind) or idiotypic antigenic determinants. A component in the IgG fraction of CM's serum was shown by one of the assay systems to also share Ind determinants with the IgA and IgM. Recent experiments have indicated that the CM IgG monoclonal protein also shares identical Ind determinants with the other two CM immunoglobulins (Ig). These results suggested that the variable regions of the heavy and light chains of the three different classes of Ig were very similar. A direct immunofluorescence assay was used in this study on bone marrow smears of patient CM obtained at two different dates to identify the cells producing these Ig. The reaction of antisera specific for Ind determinants demonstrated that most plasma cells, whether containing IgM, IgA, or IgG, stained with the anti-Ind antisera; this occurred irrespective of whether the anti-Ind antisera were generated to the patient's IgM or IgA. The reaction with isotypic antisera revealed cell populations containing either IgM, IgA, or IgG and a significant cell population containing both IgM and IgA. The common occurrence of IgM and IgA-containing cells which synthesize monoclonal Ig with shared Ind determinants provides evidence consistent with the IgM-IgA pathway of differentiation of antibody-producing cells. Further, the tendency toward decreasing numbers of IgM-staining cells with concomitant increasing numbers of IgA and IgG-containing cells suggests that the clones of cells producing these Ig were derived from a single precursor cell.     

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.     

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.     

Virus hepatitis of geese. 3. Properties of the causal agent

Characteristics of the goose hepatitis virus strain SHM 319 were studied in goose embryos and tissue cultures of avian origin. The virus was found to be resistant to both ether and sodium deoxycholate (0.25%). The growth of the virus was inhibited by 5-iodo-2-deoxyuridine. Virus infectivity was not affected by heating at 56 degrees C for 3 hours, exposure to pH 3.0, formalin (1:1000), and other inactivating chemicals. Cell culture systems of three avian species were inoculated with GVH-SHM 319. Extensive cytopathic effect was produced only in goose cell cultures. No virus replication was observed in chicken or White Pekin duck cells. Fluorescent antibody staining revealed granular nuclear staining in infected susceptible tissue cultures. May-Grunwald-Giemsa-staining of infected tissue culture cells showed formation of mainly intranuclear inclusion bodies. Millipore filtration procedures revealed a particle size less than 50 nm. Hemagglutination was not observed. Precipitating antibodies could not be detected in GHV hyperimmunized birds. Neutralization tests could not demonstrate a relationship to known viruses of waterfowl, including duck virus enteritis and duck virus hepatitis. A certain discrepancy in the neutralization test with Hungarian goose sera is discussed.     

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.     

Immunoglobulins in Fluid from Non-Keratinizing Jaw Cysts

Thirty-six fluids from non-keratinizing jaw cysts have been examined together with autologous sera by immunoelectrophoresis and double diffusion in agar or agarose gels. Except for one cyst fluid which contained electrophoretically homogeneous ('monoclonal') IgG of the χ type together with free χ chains, IgG of cyst fluid was electrophoretically heterogeneous. For the most, IgA of cyst fluid migrated more slowly than IgA of serum, whereas the IgM migrated similarly. The three immunoglobulins showed reactions of antigenic identity with the corresponding Ig classes of serum when examined with rabbit antisera against human IgG, IgA, and IgM. Fluid from a median palatine cyst contained secretory component, which showed a reaction of identity with free secretory component isolated from human saliva, and probably also IgA of the secretory type. Two cyst fluids also precipitated a component in rabbit serum.     

T lymphocytes of the human colonic mucosa: functional and phenotypic analysis.

Normal human colonic lymphocyte populations were isolated for both phenotypic analysis by double-label immunofluorescence and assessment for regulatory effects on Ig production by co-culture with responder cells from colonic mucosa and peripheral blood. Mean CD4:CD8 ratios for colonic intraepithelial lymphocytes (IEL) and lamina propria lymphocytes (LPL) were comparable to values obtained from tissue sections. IEL alone did not produce Ig in vitro and were without effect on Ig production when co-cultured with LPL. However, T-enriched LPL had a marked helper effect for T-depleted LPL. Maximal help was for IgA production, increasing with numbers of T-enriched cells. In colonic LPL T-depleted and T-enriched co-cultures, pokeweed mitogen (PWM) had no significant effect. By contrast, in co-cultures of T-enriched and T-depleted peripheral blood mononuclear cells, Ig production was PWM-dependent. In all experiments with colonic mucosal responder cells, IgG production was low. The effects of unfractionated colonic biopsy lymphocytes on T-depleted peripheral blood mononuclear cells were additive for IgM production and synergistic for IgA synthesis, although almost no IgG was produced. Moreover, PWM had helper effects for IgM, but was suppressive for IgA production. These data suggest that colonic mucosal regulatory cells reside in the lamina propria, and predominantly provide help for IgA and IgM synthesis. The data further suggest the existence of a pre-stimulated IgA-specific T helper cell population.     

Humoral response of chicken infected with the microsporidium Encephalitozoon hellem

Chicken (Gallus gallus) were used as the experimental model for study of immune response against the microsporidium Encephalitozoon hellem (Didier et al., J Inf Dis 163:617–621, 1991) infection in birds. Two-day-old chicken were infected perorally or intraperitoneally with a dose of 10⁷ spores of E. hellem. The anti-E. hellem immunoglobulin (Ig)A, IgY, and IgM antibody responses in sera and dropping sample extracts were determined by enzyme-linked immunosorbent assay. Results have shown specific antibody production in sera and intestinal secretions of infected birds. Chicken inoculated perorally developed the lowest antibody response. Microsporidian spores were not identified in the smears from cloacal swab samples of individual chicken. Intestinal segment cultures of perorally infected chicken cultivated in vitro showed the highest production of specific IgY and IgA antibodies in jejunum segments. In the further course of infection, the colon produced the highest amount of IgA, and the ileum and colon produced the highest amount of IgY.     

Immunohistochemical study of nasal mucosa in patients with selective IgA deficiency

Fifteen nasal biopsy specimens from adult patients with selective IgA deficiency were examined in a 'blind' immunohistochemical study for the presence of immunocytes producing various immunoglobulin (Ig) classes. Three groups of patients could be identified. One group had a predominance of IgG- and IgM-producing cells in their nasal mucosa, a second group revealed mainly IgG- and IgD-producing cells, and a third group had very few mucosal immunocytes. The clinical examinations showed that upper respiratory tract infections were most common in patients with few immunocytes while such infections were least common in patients with predominance of IgG and IgM immunocytes. Our results indicated that IgM, in contrast to IgD, acts as a compensatory secretory Ig in some patients with selective IgA deficiency.     

Monoclonal antibodies used to isolate IgM from chicken bile and avian sera and to detect specific IgM in chicken sera

Four hybridoma cell lines (designated M1, M10, M11, M31) were established which secrete antibody specific for chicken IgM. The specificity for the IgM heavy chain was shown by using ELISAs to screen for antibodies to IgM, IgA and IgG. Radioimmunoprecipitation tests confirmed that the four monoclonal antibodies reacted with IgM and also showed that they combined with Protein A. An immunoadsorbent was made using one (M1) of the monoclonal antibodies. IgM was purified in a single step by affinity chromatography from chicken bile and chicken, turkey and duck serum. This is the first unequivocal demonstration of chicken IgM in bile. The Ml monoclonal antibody was used in an ELISA to detect the specific chicken IgM response to the inoculation of bovine serum albumin. This anti-IgM reagent may also be used to detect the IgM response to other antigens.