Morphological and immunohistochemical analyses of soluble proteins in mucous membranes of living mouse intestines by cryotechniques.

We have performed immunohistochemical or ultrastructural analyses of living mouse small intestines using Epon blocks prepared by 'in vivo cryotechnique' (IVCT). By electron microscopy, intracellular ultrastructures of epithelial cells were well preserved in tissue areas 5-10 µm away from cryogen-contact surface tissues. Their microvilli contained dynamically waving actin filaments, and highly electron-dense organelles, such as mitochondria, were seen under the widely organized terminal web. By quick-freezing of fresh resected tissues (FT-QF), many erythrocytes were congested within blood vessels due to loss of blood pressure. By immersion-fixation (IM-DH) and perfusion-fixation (PF-DH), small vacuoles were often seen in the cytoplasm of epithelial cells, and their intercellular spaces were also dilated. Moreover, actin filament bundles were irregular in cross sections of microvilli, compared with those with IVCT. Epon-embedded thick sections were treated with sodium ethoxide, followed by antigen retrieval and immunostained for immunoglobulin A (IgA), Ig kappa light chain (Igκ), J-chain and albumin. By cryotechniques, IgA immunoreactivity was detected as tiny dot-like patterns in cytoplasm of some epithelial cells. Both J-chain and Igκ immunoreactivities were detected in the same local areas as those of IgA. By FT-QF, however, the IgA immunoreactivity was more weakly detected, compared with that with IVCT. In thick sections prepared by IM-DH and PF-DH, it was rarely observed in both plasma and epithelial cells. Another albumin was diffusely immunolocalized in extracellular matrices of mucous membranes and also within blood vessels. Thus, IVCT was useful for preservation of soluble proteins and ultrastructural analyses of dynamically changing epithelial cells of living mouse small intestines.

Quantitative peptidomics of pituitary glands from mice deficient in copper transport.

We previously described a method of quantitating levels of peptides in Cpe(fat)/Cpe(fat) mice using affinity chromatography to isolate peptide-processing intermediates and differential isotopic labeling/mass spectrometry. In the present study, we compared two different isotopic labels, acetic anhydride and succinic anhydride for detection and quantitation of peptides in wild type mice. As previously found for acetic anhydride, succinic anhydride efficiently labels all primary amines in various peptides. Of these two reagents, succinic anhydride provides better resolution between the heavy and light peaks of the labelled peptides due to a greater mass difference between the deuterated (heavy) and non-deuterated (light) form of this label (4 Da for succinate, 3 Da for acetate). Using succinic anhydride labeling, the accuracy of measuring 1:1 and 1:2 ratios of peptides in pituitary extracts was within 5% of the theoretical value for most peptides. The accuracy with succinic anhydride is comparable to the accuracy of acetic anhydride and more peptides could be detected and quantitated with succinic anhydride. The two labels were then used to examine pituitary peptides in mice with a defect in copper transport (Atp7a mice) vs wild type mice. Using succinic anhydride, 13 peptides could be detected, 12 of which matched the theoretical mass of known pituitary peptides. Five of the six peptides which contain C-terminal amide groups were significantly decreased in the Atp7a mice relative to wild type mice, whereas only one non-amidated peptide was significantly decreased in Atp7a mice. With acetic anhydride, only five peptides could be quantitated. The three peptides which contain C-terminal amide groups were decreased approximately 30% in the Atp7a mice. The selective decrease in amidated peptides in Atp7a mice is consistent with the copper-requirement of the enzyme that forms C-terminal amides.

The J chain is essential for polymeric Ig receptor-mediated epithelial transport of IgA.

Local production of secretory (S)IgA provides adaptive immunologic protection of mucosal surfaces, but SIgA is also protective when administered passively, such as in breast milk. Therefore, SIgA is a potential candidate for therapeutic administration, but its complex structure with four different polypeptide chains produced by two distinct cell types complicates recombinant production. The J chain is critical in the structure of SIgA because it is required for efficient polymerization of IgA and for the affinity of such polymers to the secretory component (SC)/polymeric (p)IgR. To better understand the role of the J chain in SIgA production, we have generated various mutant forms of the human J chain and analyzed the function of these mutants when coexpressed with IgA. We found that the C terminus of the J chain was not required for the formation of IgA polymers, but was essential for the binding of pIgA to SC. Likewise, we found that two of the intrachain disulfide bridges (Cys(13):Cys(101) and Cys(109):Cys(134)) were also required for the binding of pIgA to SC but, interestingly, not for IgA polymerization. Conversely, the last intrachain disulfide bridge (Cys(72):Cys(92)) was not essential for either of these two J chain functions. Finally, we demonstrated that the presence of only Cys(15) or Cys(69) was sufficient to support polymerization of IgA, but that these polymers were mostly noncovalently stabilized. Nevertheless, these polymers bound free SC with nearly the same affinity as pIgA containing wild-type J chain, but were transcytosed by pIgR-expressing polarized epithelial cells at a reduced efficiency.

The IgA/IgM receptor expressed on a murine B cell lymphoma is poly-Ig receptor.

T560, a mouse B lymphoma that originated in gut-associated lymphoid tissue, expresses receptors that bind dimeric IgA and IgM in a mutually inhibitory manner but have little affinity for monomeric IgA. Evidence presented in this paper indicates that the receptor is poly-Ig receptor (pIgR) known in humans and domestic cattle to bind both IgA and IgM. The evidence includes the demonstration that binding of IgM is J chain dependent, and that pIg-precipitated receptor has an appropriate Mr of 116-120 kDa and can be detected on immunoblots with specific rabbit anti-mouse pIgR. Overlapping RT-PCR performed using template mRNA from T560 cells and oligonucleotide primer pairs designed from the published sequence of mouse liver pIgR indicate that T560 cells express mRNA virtually identical with that of the epithelial cell pIgR throughout its external, transmembrane, and intracytoplasmic coding regions. Studies using mutant IgAs suggest that the Calpha2 domain of dimeric IgA is not involved in high-affinity binding to the T560 pIgR. Inasmuch as this mouse B cell pIgR binds IgM better than IgA, it is similar to human pIgR and differs from rat, mouse, and rabbit epithelial cell pIgRs that bind IgA but not IgM. Possible explanations for this difference are discussed. All clones of T560 contain some cells that spontaneously secrete both IgG2a and IgA, but all of the IgA recoverable from the medium and from cell lysates is monomeric; it cannot be converted to secretory IgA by T560 cells.

Decreased vaginal disease in J-chain-deficient mice following herpes simplex type 2 genital infection.

J-chain-deficient (Jch(-/-)) mice were used to study the role of polymeric IgA (pIgA) in primary disease and protective immunity following genital herpes simplex type 2 (HSV-2) infection. Vaginal IgA in the Jch(-/-) mice was composed primarily of monomeric IgA and was not associated with secretory component (SC). In contrast, vaginal IgA in wild-type (WT) mice was predominantly polymeric and bound to SC. Following HSV-2 genital infection, the Jch(-/-) mice consistently exhibited fewer vaginal symptoms (P = 0.010) and mortality (P = 0.075) than did the WT mice. The variation in disease expression could not be explained by differences in local viral replication, since titers in vaginal wash fluid were comparable. To assess the effect of J chain deficiency on protective immunity, WT and Jch(-/-) mice were immunized intravaginally with attenuated HSV-2, challenged intravaginally with wild-type virus 5 weeks later, and evaluated for vaginal infection and neurological disease. Although the Jch(-/-) mice had reduced vaginal HSV-specific IgA and IgG levels following immunization, both WT and Jch(-/-) mice were protected from symptoms following wild-type virus challenge. We conclude that pIgA is not required for protective immunity against genital HSV-2 disease and that J chain deficiency offers some protection against symptoms following primary HSV-2 genital infection.

Regulation of the formation and external transport of secretory immunoglobulins.

Secretory IgA (SIgA) is the best defined effector component of the mucosal immune system. Generation of SIgA and secretory IgM (SIgM) in exocrine glands and mucous membranes depends on a fascinating cooperation between local plasma cells that produce polymeric IgA (pIgA, mainly dimers and some larger polymers) and pentameric IgM, and secretory epithelial cells that express the polymeric Ig receptor (pIgR)--also known as transmembrane secretory component. After release from the local plasma cells and diffusion through the stroma, pIgA and pentameric IgM become readily bound to pIgR, and are then actively transported across secretory epithelial cells for extrusion into external secretions after cleavage of pIgR. Much knowledge has recently been obtained at the molecular level about the regulation of pIgR-mediated transport of antibodies. This mechanism is of considerable biological interest because SIgA and SIgM form the first line of specific immunological defense against infectious agents and other harmful substances that may enter the body through the mucosae.

Antibody against the human J chain inhibits polymeric Ig receptor-mediated biliary and epithelial transport of human polymeric IgA.

To emphasize the requirement for a J chain in native polymeric immunoglobulins for their selective transport into exocrine secretions, IgG, purified from two different antisera specific for the human J chain, was shown to: (i) bind in vitro to human polymeric IgA (pIgA) by density gradient ultracentrifugation; (ii) inhibit binding in vitro of rat secretory component to human pIgA; (iii) inhibit hepatic transport of human pIgA into rat bile in vivo; and (iv) inhibit apical transcytosis of pIgA in vitro by polarized human polymeric immunoglobulin receptor (pIgR)-expressing Madin-Darby canine kidney cells. Inhibition of biliary transport increased with the molar ratio of anti-J chain antibodies against pIgA and their incubation time. Anti-J chain F(ab')2 and Fab fragments also inhibited biliary transport, excluding a role for phagocytic clearance or excessive size of the immune complexes. Anti-human-Fc alpha Fab, bound to human pIgA in complexes of larger size than those with anti-J chain Fab, did not inhibit biliary transport of human pIgA. Propionic acid-denatured human pIgA, although containing J chains, was very poorly transported into rat bile. Altogether, our data strongly support, now also by in vivo experiments, the crucial role of the J chain of native pIgA in its selective pIgR-mediated transport into secretions, as suggested long ago by in vitro data only. Recent data on J chain-knockout mice, with low IgA levels in bile and feces, cannot explain the role of the J chain in contributing to the secretory component/pIgR-binding site of normal pIgA, but otherwise agree with our study.

Mechanisms of heterosubtypic immunity to lethal influenza A virus infection in fully immunocompetent, T cell-depleted, beta2-microglobulin-deficient, and J chain-deficient mice.

Immunity that is cross-protective between different influenza A virus subtypes (termed heterosubtypic immunity) can be demonstrated readily in some animals but only rarely in humans. Induction of heterosubtypic immunity in humans by vaccines would provide public health benefit, perhaps offering some protection against pandemics or other new influenza A strains. Therefore, we studied mechanisms mediating heterosubtypic immunity in mice. Immunization with either A/H1N1 or A/H3N2 virus protected mice against mortality following heterosubtypic challenge while providing modest reductions in lung virus titers. No cross-protection was seen with distantly related type B influenza virus. Depletion of CD4+ or CD8+ T cells or both around the time of challenge had no significant effect on survival, indicating that these cells are not required at the effector stage. beta2-microglobulin knockout mice could be protected readily against heterosubtypic challenge, confirming that class I-restricted T cells are not required. In beta2-microglobulin -/- mice, depletion of CD4+ T cells partially abrogated heterosubtypic immunity, showing that they play a role in these mice. Passive transfer of Abs to naive recipients protected against subsequent challenge with homologous but not heterosubtypic virus. Because a role for secretory Abs has been suggested, we studied dependence on the J chain, which is required for polymeric Ig receptor-mediated IgA transport. J chain knockout mice were readily protected by heterosubtypic immunity, indicating that polymeric Ig receptor-mediated transport is not required. Better understanding of heterosubtypic immunity should be valuable in analyzing new vaccines, including peptide and DNA vaccines, intended to induce broadly cross-reactive immunity.

[Molecular aspects of secretory IgA (S-IgA) in gut-associated lymphoid tissues].

Secretory IgA, which plays an important role in the defense of the exocrine tissue, is composed of a polymeric IgA, joining (J) chain and secretory component (SC). Polymeric IgA and J chain are produced by plasma cells and SC by glandular epithelial cells. We here described the molecular aspects of J chain and SC. Study of the J chain has been confined to vertebrates which produce immunoglobulin (Ig) because the function of J chain is considered to be a polymerization of Ig. Recent molecular studies indicate that the role of J chain has been questioned. The J chain is expressed in invertebrates, as well as, representative species of vertebrates and that J chain is a primitive polypeptide that arose before the evolution of Ig molecules. SC is a 80 kDa glycoprotein functioning as a receptor for J chain-containing polymeric Ig. The expression of SC is regulated by various inflammatory cytokines such as, IL-1, IL-4, IL-6, IL-8, IFN-gamma and TNF-alpha, suggesting SC upregulation in vivo in inflammatory conditions. The human SC cDNA analysis reveals that it consisted of 11 exons with no functional TATA-box or CCAAT-box in the putative promoter region. Further upstream, there are several interesting motifs such as NF-kB and IFN-gamma response element, suggesting possible regulation of SC by cytokines through cellular signal transduction pathways.

Ultrastructural localizations of J chains in the chicken bursa of Fabricius at different stages of development.

Before and after hatching, J-chain positive cells (JPC) were observed by immunoelectron microscopy in the chicken bursa of Fabricius. JPC were mostly lymphocytes, but epithelial cells were also detected as JPC. During the embryonic stage, J chains were mostly associated as patches with surface membranes. Furthermore, there was a diffuse localization in the cytoplasm. After hatching, J chains showed a similar subcellular localization as was seen before hatching. However, J chains were frequently detected in the cytoplasm, and rarely on the surface membranes after hatching. Staining intensities by corresponding antisera were stronger in the hatched chickens than in embryos. From these findings one may conclude that J chains are synthesized even at an early stage of B cell differentiation during embryonic life and are continuously produced at the later differentiation stages of B-cell lineage. The increased amounts of J chains estimated by staining intensity seem to coincide with B cell maturation and may correlate with signalling of IgM synthesis.

Secretion of immunoglobulin M assembly intermediates in the presence of reducing agents.

There are several demonstrations that misfolded or unassembled proteins are not transported along the secretory pathway, but are retained intracellularly, generally in the endoplasmic reticulum. For instance, B lymphocytes synthesize but do not secrete IgM, and only the polymeric form of IgM is secreted by plasma cells. The C-terminal cysteine of the mu heavy chain of secreted IgM (residue 575) is involved in the intracellular retention of unpolymerized IgM subunits. Here we report that the addition of reducing agents to the culture medium, at concentrations which do not affect cell viability, terminal glycosylation, or retention of proteins in the endoplasmic reticulum through the KDEL mechanism, induces secretion of IgM assembly intermediates by both B and plasma cells. Free joining (J) chains, which are not normally secreted by plasma cells unless as part of IgM or IgA, are also secreted in the presence of reducing agents. We propose a role for free thiol groups in preventing the unhindered transport of proteins through the secretory pathway. Under the scheme, assembly intermediates interact through their thiol groups between themselves and/or with unknown proteins of the endoplasmic reticulum. Such interactions may be prevented by altering the intracellular redox potential or by site-directed mutagenesis of the relevant cysteine residue(s).

The biological effects of IgM hexamer formation.

The inducible B cell lymphoma, CH12, and its in vitro adapted subclone, CH12-LBK, produce immunoglobulins of identical sequence, specificity and isotype, with equivalent affinities for the hapten trimethyl ammonium. However, the hemolytic efficiencies of the antibody secreted by the two cell lines are quite different. Antibody preparations from lipopolysaccharide-stimulated CH12 cells lyse erythrocytes six- to ten times more effectively than antibody preparations of the same concentration from CH12-LBK cells. Both cell lines secrete polymeric IgM, but while CH12-LBK cells secrete predominantly the canonical pentameric IgM, CH12 cells secrete a mixture of pentamers and hexamers. High-efficiency complement-dependent cytolysis is associated with hexameric IgM, which has a specific activity that is approximately twenty times higher than that of the pentameric form. J chain protein is found in the secreted IgM of both cell lines, but is associated only with the pentameric IgM and not with the hexameric form, nor with any intermediate polymers smaller than a pentamer. A deficit in, or the inaccessibility of, J chain protein appears to facilitate hexamer formation. These experiments confirm previously published data showing that J chain is not necessary either for assembly or secretion of polymeric IgM, and suggest instead that J chain may be important in regulating the lytic efficiency of polymeric IgM by controlling the IgM pentamer/hexamer ratio. The experiments further suggest a mechanism, in addition to isotype switching and somatic mutation, by which the biological efficiency of antibodies from a single clone of B cells can be regulated.

The rabbit kappa 1 b5 immunoglobulin gene: another J region gene cluster with only one functional J gene segment?

Our previous analysis of an immunoglobulin gene encoding the rabbit kappa chain of b4 allotype revealed that of the five J-like sequences in the J kappa cluster of this gene, only one, J2, appeared to be functional. This unusual ratio of J pseudogenes to functional J genes is unique among all J clusters of light and heavy chain genes of all species examined to date, including the cluster from the rabbit kappa 2 isotype, and must have consequences for diversity generation of b4 immunoglobulins. The fact that the only two known b5 J kappa sequences are different from the functional J2 of the b4 allotype prompted investigation of the b5 J kappa cluster to determine whether it resembled the b4 cluster, or the more typical mouse or human J kappa clusters. Our analysis of the b5 gene reveals a J kappa cluster strikingly similar to that of b4; apparent defects occur in all J sequences except J2. Although J2 is apparently functional, it differs from the J2 of the b4 locus by four nucleotide and three amino acid substitutions. The unusually high degree of sequence similarity previously observed between the b4 and b5 loci in the noncoding (vs the coding) regions extends through the newly sequenced DNA segment and remains an enigma.

[The role of IgA in allergic disease]. / Ruolo delle IgA nelle allergopatie.

Within the framework of immunological mechanisms involved in allergic forms, and view of the fact that immunoglobulins represent one of the cardinal points thereof, the attention is focused on the IgA, first getting a deeper insight into the current knowledge of their structure and localization, which has recently been added to with some important details. The models set forth are aimed to show the molecular organization of monomeric and polymeric IgA, and the functional significance of their different distribution in secretory and serous tissues, with particular stress on the action displayed by IgA specific bacterial proteases. Thus the problem of IgA synthesis and secretion, analyzing the model of formation of secretory IgA, and the defensive role they play in protecting the mucous surfaces, is tackled. Lastly, within the framework of IgA-specific immunodeficiencies, the pathogenetic mechanisms of the selective hypo-IgA, the variability of the IgA selective defect, and the pathology connected therewith, are examined with particular stress on the relationships between IgA and allergopathies along with the latest immunotherapy trends, understood as a supply of the lacking immunoglobulin class.

Further evidence for a role of secretory component (SC) and J chain in the glandular transport of IgA.

These studies support the transport model depicted in Figure 3: Incorporation of J chains into dimeric IgA induces a configurational fit allowing complexing of IgA with SC available in the plasma membrane of serous secretory epithelial cells. This complexing on the surface of the cell stimulates pinocytosis, and the completed secretory IgA molecules are transported in cytoplasmic vesicles to the gland lumen. The same transport model can be applied for the external translocation of IgM.