J-chain expression in human cells producing IgG subclasses

Previous studies have demonstrated that J chain is expressed not only in cells that produce polymeric immunoglobulins, but also in those engaged in synthesis of monomers including IgG and IgD. The presence of J chain in these cells suggested that its role may not be restricted to the formation of polymers. For the present study, fluorochrome-labeled polyclonal anti-J-chain and monoclonal antibodies to IgG subclasses were used to determine the distribution of J chain in IgG plasma cells from normal human tissues and from pokeweed mitogen (PWM)-stimulated human peripheral blood lymphocytes. The results indicate that J chain is not equally distributed among cells producing different IgG subclasses. The percentages of PWM-stimulated cells containing J chain were: 22 +/- 5 (SE) for IgG1, 49 +/- 6 for IgG2, 17 +/- 7 for IgG3, and 64 +/- 11 for IgG4. Examination of sections of various human lymphoid tissues revealed that the frequency of IgG cells that coexpressed J chain was lower than that observed in the PWM system and displayed variable distribution among IgG subclasses. The frequency of J-chain expression in IgG-producing cells may be related to the degree of cellular maturation and may differ according to the origin of cells.     

The carboxyl-terminal domains of IgA and IgM direct isotype-specific polymerization and interaction with the polymeric immunoglobulin receptor

Mucosal surfaces are protected by polymeric immunoglobulins that are transported across the epithelium by the polymeric immunoglobulin receptor (pIgR). Only polymeric IgA and IgM containing a small polypeptide called the "joining" (J) chain can bind to the pIgR. J chain-positive IgA consists of dimers, and some larger polymers, whereas only IgM pentamers incorporate the J chain. We made domain swap chimeras between human IgA1 and IgM and found that the COOH-terminal domains of the heavy chains (Calpha3 and Cmu4, respectively) dictated the size of the polymers formed and also which polymers incorporated the J chain. We also showed that chimeric IgM molecules engineered to contain Calpha3 were able to bind the rabbit pIgR. Since the rabbit pIgR normally does not bind IgM, these results suggest that the COOH-terminal domain of the polymeric immunoglobulins is primarily responsible for interaction with the pIgR. Finally, we made a novel chimeric IgA immunoglobulin, containing the terminal domain from IgM. This recombinant molecule formed J chain-containing pentamers that could, like IgA, efficiently form covalent complexes with the human pIgR ectodomain, known as secretory component.     

Biosynthesis of immunoglobulin A (IgA) and immunoglobulin M (IgM). Control of polymerization by J chain

Cell suspensions of mouse plasma-cell tumours secreting IgA (immunoglobulin A) and IgM (immunoglobulin M) were incubated with radioactive leucine for various periods of time. The secreted immunoglobulins were precipitated from the culture medium with specific rabbit antisera to determine the relative distribution of radioactivity among the different molecular species, and to estimate the fraction of total radioactivity in the J chain. For IgM-secreting cells there is a balanced synthesis of 7S subunits and J chains, and the secreted product is uniformly assembled to the pentamer. In cells secreting IgA, however, the results demonstrate that the pool of intracellular J chain is less than the intracellular IgA pool. The concentration of J chain is therefore limiting and is less than the requirement for complete polymerization. The major factor that determines whether an intracellular monomer is secreted as such or is polymerized with the addition of J chain is therefore the amount of intracellular J chain. When this is limiting, as it is in cells secreting IgA, then monomer will be secreted.     

Partial amino acid sequence of a rabbit immunoglobulin light chain of allotype b5

The amino acid sequence of a rabbit immunoglobulin light chain of allotype b5 has been nearly completed. A comparison of its structure with that of light chains of allotypes b4, b6, and b9 confirms that the constant regions of these various kappa chains differ by 20-35%. The substitutions are clustered in parts of the second half of the chain, and the b5 form bears more resemblance to the b6 chain than to any other, in good agreement with previous serological data. The analysis of the variable region reveals the existence of certain allotype-associated residues which have also been reported in other b5 chains, but not in proteins of the other allotypes. An examination of the rabbit light chain sequences between positions 96 and 107 suggests that this portion of the chain may be encoded separately by a joining "J" DNA segment, as has been described previously for murine and human immunoglobulins. In the rabbit, however, these J kappa regions appear to differ from one allotype to another. Together with the extensive variations of the constant regions, these data suggest that the rabbit kappa gene organization more closely resembles the murine gamma system (four different C gamma genes each flanked by its J segment) than the murine kappa system (a single C kappa gene).     

Immunocytochemical identification and localization of immunoglobulin A within Paneth cells of the rat small intestine.

Light microscopic immunocytochemistry was used to identify Paneth cells by their lysozyme content and to detect immunoglobulin antigens within a subpopulation of these cells. Antisera specific for the heavy chains of rat or human immunoglobulin A and for immunoglobulin light chain antigens produced specific staining of rat Paneth cells. The distribution of immunoglobulin staining varied between adjacent Paneth cells in the same crypt and between Paneth cells in adjacent crypts, as well as between Paneth cell populations of different animals. No staining of rat Paneth cells was detected using antisera specific for the heavy chain of immunoglobulins G or M. The specific staining of Paneth cells for immunoglobulin A and light chain antigens was blocked by absorption of each antiserum with its respective purified antigen. Absorption of these antisera with purified rat lysozyme did not affect staining and thereby eliminated the possibility of immunologic cross-reactivity between lysozyme and immunoglobulin antigens. It is suggested, in light of current concepts of Paneth cell function, that the immunoglobulin staining of Paneth cells may reflect their ability to phagocytize immunoglobulin A-coated microorganisms or immune complexes containing immunoglobulin A.     

Biosynthesis of immunoglobulin A (IgA) and immunoglobulin M (IgM). Requirement for J chain and a disulphide-exchanging enzyme for polymerization

Mouse myeloma cells secreting 19S IgM (immunoglobulin M) (MOPC 104E and TEPC 183) or monomer and polymer IgA (immunoglobulin A) (MOPC 315) were incubated with radioactive leucine and the intracellular and secreted immunoglobulins and immunoglobulin subunits were prepared by preparative sucrose-density-gradient centrifugation. Samples were reduced in the presence or absence of isolated J chain, passed over Sephadex G-25 and then incubated at 37 degrees C for 30min with or without a source of disulphide-interchange enzyme. The extent of reassembly of reduced subunits was then evaluated by electrophoresis in polyacrylamide gels. Provided that J chain and the disulphide-interchange enzyme were supplied, both IgM and IgA could be assembled from their respective subunits, obtained by reductive cleavage of polymeric forms. Under similar conditions, assembly of polymeric forms from intracellular or secreted 7S monomer subunits also occurred. Under these conditions polymerization was total, there being no residue of the monomeric form. Reassembly did not occur in the absence of either J chain or the enzyme. All of the J chain released from IgM by reductive cleavage was incorporated back into the reassembled polymer. The J chain is therefore likely to be an essential structural requirement for polymeric immunoglobulins. A variety of controls ruled out non-specific interactions, and further suggested that the amino acid sequence of polypeptide chains determines the specificity of polymerization. The fact that intracellular IgA and IgM monomer subunits known to be deficient in galactose and fucose can be completely polymerized suggests that the addition of carbohydrate does not control polymerization.     

Upregulated expression of kappa light chain by Epstein-Barr virus encoded latent membrane protein 1 in nasopharyngeal carcinoma cells via NF-kappaB and AP-1 pathways

B lymphocytes are generally considered to be the only source of immunoglobulins. However, increasing evidence revealed that some human epithelial cancer cell lines, including nasopharyngeal carcinoma (NPC) cell lines, expressed immunoglobulins. Moreover, we previously found that expression of kappa light chain in NPC cells could be upregulated by EBV-encoded latent membrane protein 1 (LMP1). Here, Western blot and flow cytometric analysis of intracellular kappa staining indicated that upregulation of the expression of kappa was inhibited by using LMP1-targeted DNAzyme and that Bay11-7082 and SP600125, inhibitors of JNK and NF-kappaB, respectively, inhibited LMP1-augmented kappa light chain expression in NPC cells. LMP1-positive NPC cells expressing the dominant-negative mutant of IkappaBalpha (DNMIkappaBalpha) or of c-Jun (TAM67) exhibited significantly decreasing kappa production compared with their parental cells. These results suggest that LMP1 elevated kappa light chain through activation of the NF-kappaB and AP-1 signaling pathways. The present study provided some hints of possible mechanisms by which human cancer cells of epithelial origin produced immunoglobulins.     

Murine plasma cells secreting more than one class of immunoglobulin. VI. Secretion of completely assembled IgG2b and IgA molecules with segregated heavy chains and free light chains by spontaneous myeloma SAMM 368 in culture.

The antigenic and molecular characteristics of the two immunoglobulins secreted by a single cell line of plasmacytoma SAMM 368 were analyzed by polyacrylamide gel electrophoresis of biosynthesized proteins. Adapted to continuous in vitro cultivation, this BALB/c plasmacytoma secretes at least 98% of its heavy chains as components of fully assembled and isotypically uniform IgG2b and IgA molecules. The IgA is secreted as monomers, dimers, and multimers with chemical properties typical of BALB/c myeloma IgA including disulfide bonded J chain and noncovalently bonded light chains. The noncovalently bonded light chains are monomers rather than dimers. Free light chains are also secreted. The ability to segregate heavy chains is attributed either to chemical, enzymatic, or compartmental regulatory factors operating within these plasma cells.     

Immunological investigation of the tissue specificity of the J peptide of human secretory immunoglobulins

The content of the J peptide of secretory polymeric immunoglobulins can significantly change during various pathologies, reflecting the state of the adaptive immune system. In this study, the content of J peptide was determined in various tissues of healthy people. These results can be used as basic data for investigations of the changes in the content of J peptide during different pathologies.     

Immunohistochemical studies on various aspects of glandular immunoglobulin transport in man

Summary Gland-associated immunocyte populations have been characterized in human tissue specimens from which extracellular immunoglobulins have been removed by saline extraction. There is a striking preponderance of IgA-producing immunocytes adjacent to glands of the gastro-intestinal and respiratory tracts, in minor and major salivary glands, and in lactating mammary glands. Immunohistochemically, these cells have been found to contain dimeric IgA with incorporated J chain. Despite this local IgA production, immunohistochemical tests on alcohol-fixed specimens demonstrate that the glandular stroma is normally permeated predominantly by IgG, most of which is obviously serum-derived. However, the serous glandular cells selectively transmit dimeric IgA, which appears along their lateral borders and apically in the cytoplasm, whereas the epithelial occurrence of IgG is less conspicuous and is restricted to the interstices.The same epithelial cells produce a glycoprotein called the secretory component (SC) which exhibits specific affinity for J chain-containing dimeric IgA and pentameric IgM. In saline-extracted tissue, IgA, but IgG, is retained regularly along the borders of SC-producing cells; this probably reflects complexing between locally formed IgA and SC in the epithelial cell membranes. SC apparently functions as a glandular receptor for dimeric IgA which thus most likely enters the epithelial cells by adsorptive pinocytosis. After covalent stabilization, the IgA-SC complexes are extruded to the gland lumen. Immunohistochemically the Golgi zone has been found to contain free SC but no IgA, whereas SC occurring more apically in the epithelial cell exhibits characteristics of being IgA-associated. Pentameric IgM is handled by the glands in a way similar to dimeric IgA, but local synthesis of IgM is normally negligible, except in the gut.     

Molecular anatomy of the antibody binding site

The binding region of immunoglobulins, which includes the portion of the molecule having the most variability in its amino acid sequence, is shown to have a surprisingly constant structure that can be characterized in terms of a simple, well-defined model. The binding region is composed of the antigen combining site plus its immediate vicinity and arises by noncovalent association of the light and heavy chain variable domains (VL and VH, respectively). The antigen combining site itself consists of six polypeptide chain segments ("hypervariable loops") which comprise some 80 amino acid residues and are attached to a framework of VL and VH beta-sheet bilayers. Having analyzed refined x-ray crystallographic coordinates for three antigen-binding fragments (Fab KOL (Marquart, M., Deisenhofer, J., and Huber, R. (1980) J. Mol. Biol. 141, 369-391), MCPC 603 (Segal, D., Padlan, E. A., Cohen, G. H., Rudikoff, S., Potter, M., and Davies, D. R. (1974) Proc. Natl. Acad. Sci. U. S. A. 71, 4298-4302), and NEW (Saul, F. A., Amzel, L. M., and Poljak, R. J. (1978) J. Biol. Chem. 253, 585-597] we use the results to introduce a general model for the VL-VH interface forming the binding region. The region consists of two closely packed beta-sheets, and its geometry corresponds to a 9-stranded, cylindrical barrel of average radius 0.84 nm with an average angle of -53 degrees between its two constituent beta-sheets. The barrel forms the bottom and sides of the antigen combining site. The model demonstrates that the structural variability of the binding region is considerably less than was thought previously. Amino acid residues which are part of the domain-domain interface and appear not to be accessible to solvent or antigen contribute to antibody specificity.     

Direct evidence that J chain regulates the polymeric structure of IgM in antibody-secreting B cells.

IgM is secreted in two functional polymeric forms. Secreted IgM was originally thought to be exclusively a pentameric molecule containing J (joining) chain, but many B cells also secrete hexameric IgM lacking J chain. Hexameric IgM may play an important role in the immune system, since it is up to 20 times more active than pentameric IgM in initiating the complement cascade. The predominant polymeric form of IgM secreted by B cell lines, either pentameric or hexameric, correlates with the concentration of J chain present during polymerization, and cells that express high levels of J chain secrete mostly IgM pentamers. The B cell lymphoma WEHI-231 does not express J chain, and the majority of its secreted IgM is polymerized as hexamers. When a J chain-encoding cDNA was expressed in these cells, the secreted IgM was found to be almost exclusively pentameric. However, although the expression of J chain dramatically altered the phenotype of the IgM secreted by these cells, it had little effect on their secretory rate. We conclude that J chain regulates the structure and function of the IgM polymers secreted by B cells, but it is not necessary for either IgM polymerization or secretion.     

Structural requirements for polymeric immunoglobulin assembly and association with J chain

Both IgM and IgA exist as polymeric immunoglobulins. IgM is assembled into pentamers with J chain and hexamers lacking J chain. In contrast, polymeric IgA exists mostly as dimers with J chain. Both IgM and IgA possess an 18-amino acid extension of the C terminus (the tail-piece (tp)) that participates in polymerization through a penultimate cysteine residue. The IgM (mutp) and IgA (alphatp) tail-pieces differ at seven amino acid positions. However, the tail-pieces by themselves do not determine the extent of polymerization. We now show that the restriction of polymerization to dimers requires both C(alpha)3 and alphatp and that more efficient dimer assembly occurs when C(alpha)2 is also present; the dimers contain J chain. Formation of pentamers containing J chain requires C(mu)3, C(mu)4, and the mutp. IgM-alphatp is present mainly as hexamers lacking J chain, and mumugammamu-utp forms tetramers and hexamers lacking J chain, whereas IgA-mutp is present as high order polymers containing J chain. In addition, there is heterogeneous processing of the N-linked carbohydrate on IgA-mutp, with some remaining in the high mannose state. These data suggest that in addition to the tail-piece, structural motifs in the constant region domains are critical for polymer assembly and J chain incorporation.     

Binding of different antigen-enzyme and antibody-enzyme conjugates by intracellular antibodies in cytoplasm and Golgi complex of plasma cells

Summary Intracellular immunoglobulins in plasma cells were characterized by antigen-enzyme conjugates and anti-immunoglobulin antibody-enzyme conjugates applied in a double immunocytochemical approach. After their assemblage, immunoglobulins in the cytoplasm of anti-TNP antibody producing plasma cells can be demonstrated both by TNP-enzyme conjugates and by anti-immunoglobulin ( or chain specific) antibody-enzyme conjugates. Once arrived in the Golgi complex (GC) detection with TNP-enzyme conjugates remains possible, but anti-immunoglobulin antibody-enzyme conjugates did not bind to a detectable degree. Similar results were obtained in experiments where immunoglobulin-enzyme conjugates were used both as an antigen-enzyme conjugate and as an antibody-enzyme conjugate.     

Heterologous protection induced by the inner capsid proteins of rotavirus requires transcytosis of mucosal immunoglobulins

Protective immunization against rotavirus (RV) can be achieved with heterologous RV, i.e., virus isolated from another species, and with heterologous inner core VP2 and VP6 proteins assembled as virus-like particles (VLP). Although the antigenically conserved VP6 protein does not induce in vitro-neutralizing antibodies, it may, however, elicit immunoglobulins (Ig) involved in heterologous protection, as some IgA against VP6 prevent RV infection in a backpack mouse model. The protective role of Ig directed to the RV inner core proteins VP2 and VP6 was investigated in J-chain-deficient mice (J chain(-/-)), which have a defect in the polymeric Ig receptor (pIgR)-mediated transcytosis of IgA and IgM. J chain(-/-) mice and wild-type (WT) mice were intranasally vaccinated with bovine RV-derived VLP2/6 and then challenged with highly infectious murine ECw RV. Whereas WT mice were totally protected, immunized J chain(-/-) mice shed RV for several days. In addition, na?ve J chain(-/-) mice exhibited a 2-day delay in clearing RV compared with WT mice. The immunized J chain(-/-) mice displayed unaltered VLP2/6-specific B-cell numbers in spleen and in mesenteric nodes and similar levels of serum anti-VLP2/6 Ig, confirming that the adaptive B-cell response is preserved in J chain(-/-) mice. These results indicate that J-chain-mediated transcytosis of Ig participates in the clearance of RV and that epithelial pIgR-mediated transport of Ig is involved in the heterologous protection induced by VLP2/6.     

Decrease in expression of human J-chain in lung squamous cell cancer and adenocarcinoma

Secretory polymeric immunoglobulins (IgA dimers and IgM pentamers) are unique in that, apart from L- and H-chains, they contain J-chains responsible for their oligomerization. These antibodies are part of the local adaptive immune system acting on mucosa membranes of the respiratory and digestive systems as the first protection barrier to potential infectious agents. Secretory polymeric immunoglobulins are produced by highly specific B-cells and actively transported to the surface of mucosa membrane through epithelium cells. Therefore, their synthesis and J-chain content are dependent upon epithelium translocation function and condition that are markedly affected by tumorous transformation. Here, we used RT-PCR and immunoblotting to study of the J-chain content and its mRNA expression level in normal and tumorous tissues in lung squamous cell cancer and adenocarcinoma at various stages of disease progression.     

Potentiation of transmembrane signaling by cross-linking of antibodies against the beta chain of the T cell antigen receptor of JURKAT T cells.

Three monoclonal antibodies (mAb) 2D1, 3B9, and 3B12 were produced by immunizing BALB/c mice with JURKAT cells. These mAb induce comodulation of the TCR/CD3 complex expressed on JURKAT cells, but do not react with the CD3- JURKAT variant, J.RT3.T3.1. Immunoprecipitation studies with detergent-solubilized JURKAT cell lystes indicate that these mAb react with proteins having characteristics of the TCR molecules. Their low reactivity with peripheral blood mononuclear cells (PBMC) and lack of reactivity with other CD3+ T cell lines suggest that they may be anti-idiotypic mAb. Results from binding inhibition assays, reactivity with PBMC, and generation of transmembrane signals suggest that these three anti-TCR mAb recognized different epitopes on the TCR beta chain of JURKAT cells. Although the three mAb are capable of inducing the production of inositol phosphates and cytosolic free Ca2+ increase in JURKAT cells, their stimulatory capacities vary and are lower than that observed by anti-CD3 antibody (OKT3) stimulation. However, crosslinking these mAb with rabbit antimouse immunoglobulins potentiates the stimulatory response to comparable levels induced by OKT3. These mAb could be useful as tools to study V beta 8+ T cells in relation to antigen-specific activation.     

DNA content analysis of peripheral blood B-lymphocytes in plasma cell malignancies

A double fluorescence assay has been employed for the detection of cell surface and/or cytoplasmic immunoglobulins (Ig) and the measurement of nuclear DNA content in the same cell. Following staining for Ig by means of FITC conjugated antibodies directed against heavy or light chains, cell suspensions or cytospin preparations were ethanol fixed and stained with a propidium iodide-RNAse solution. In this way, the cytometric DNA content of circulating B-lymphocytes was analyzed in three patients suffering from plasma cell malignancies with an excess of peripheral blood B-lymphocytes and evidence of aneuploid bone marrow plasma cells. Aneuploid circulating B-lymphocytes with the same DNA stem-line as bone marrow plasma cells were found in two patients with advanced disease but not in the only one we studied at presentation. Aneuploid lymphocytes had surface immunoglobulins bearing the same light chain as the M-protein. In addition, a significant percentage (23%) of cells lacking either surface or cytoplasmic immunoglobulins proved to be aneuploid in plasma cell leukemia. Nuclear DNA measurement combined with surface or cytoplasmic marker analysis appears to be a reliable method for studying neoplastic lymphoid precursor cells in plasma cell malignancies.     

Intermolecular disulfide bonding in IgM: effects of replacing cysteine residues in the mu heavy chain.

The conventional model of polymeric IgM depicts a unique structure in which the mu heavy chains and J chain are joined by well defined disulfide bonds involving cysteine residues at positions 337, 414 and 575 of the mu chain. To test this model, we have used site directed mutagenesis to produce IgM in which these cysteines have been replaced by serine. In each case the single mutants were able to assemble polymeric IgM, which was analyzed for its size, morphology, J chain content and activity in complement dependent cytolysis. Whereas normal polymeric IgM is composed predominantly of pentameric and hexameric molecules, the mutant IgM-Ser414 is covalently assembled as pentamers and smaller forms; IgM-Ser575 is assembled as covalent hexamers. IgM-Ser337 appears to include the same pentameric and hexameric forms as normal IgM except that, unlike normal polymeric IgM, most pentameric/hexameric IgM-Ser337 is not covalently assembled. J chain is present in polymeric IgM-Ser337 but absent in polymeric IgM-Ser414 and IgM-Ser575. IgM-Ser414 is defective in activating the classical pathway of complement dependent cytolysis. Our observations are consistent with models in which the covalent linkages between mu chains are mediated by disulfide bonded Cys337-Cys337, Cys414-Cys414 and Cys575-Cys575 but indicate that the arrangement of these Cys-Cys pairs in series and in parallel varies among and within IgM molecules.(ABSTRACT TRUNCATED AT 250 WORDS)