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.     

IgM expressed by leukemic CD5(+) B cells binds mouse immunoglobulin light chain

Mouse immunoglobulin (Ig) molecules have previously been shown to bind to the surface of CD5(+) B cells from patients with B-cell chronic lymphocytic leukemia (B-CLL). The results indicated that surface IgM was involved in the interaction and suggested the phenomenon was an example of the polyreactive binding capacity of the surface Ig (sIg) expressed by these malignant cells. This article describes the further characterization of the interaction between human IgM and mouse Ig molecules and subunits. Mouse Ig molecules of both kappa and lambda light chain classes bound to the B-CLL cell surface. The dissociation constant for the interaction of mouse IgG1 (K121) with the B-CLL cell surface was 3.6 x 10(-7) M. To confirm the involvement of the human IgM expressed by the B-CLL cells in the interaction, the malignant cells were stimulated in vitro to induce secretion of human IgM. Enzyme immunoassay was used to show that secreted human IgM bound to intact mouse Ig, as occurred with the cell surface analysis. The mouse Ig epitope recognized by the purified secreted human IgM was shown by Western blot analysis to be located on the light chain of the mouse Ig molecule and to be conformationally dependent. K121 light chain was cloned and expressed in E. coli and the recombinant light chain bound to the surface of CLL B cells. The results confirm that human IgM is the reactive ligand in the interaction with mouse Ig and indicate that the interaction of polyreactive IgM with mouse IgG occurs via the light chain component of IgG.     

Polymeric immunoglobulin M is secreted by transfectants of non-lymphoid cells in the absence of immunoglobulin J chain.

Plasmids were constructed in which expression of genes encoding the heavy and light chains of a hapten-specific IgM antibody is under control of a heat shock promoter. Glioma, phaeochromocytoma and other non-lymphoid cell lines transfected with the plasmids were able to process and secrete immunoglobulin following heat induction. The glioma transfectants were studied in detail and were shown to secrete polymeric IgM in a yield similar to that obtained with a plasmacytoma. However, the glioma IgM was not associated with J chain and was largely composed of pentamers and hexamers. Thus, neither J chain nor other lymphoid-specific proteins are required for assembly and secretion of polymeric IgM although the absence of J chain may encourage hexamer formation.     

Evidence for an IgD homologue on chicken lymphocytes

Chicken lymphocyte membrane immunoglobulins (Ig), were precipitated with mouse monoclonal antibodies specific for heavy and light chain isotypes and analyzed by polyacrylamide gel electrophoresis. Very little or no membrane-bound IgG and IgA was detected. After sequential precipitation and removal of IgM reactive with any of three monoclonal anti-mu antibodies, anti-light chain antibody precipitated residual Ig with a relative electrophoretic mobility similar to that of IgM. Under reducing conditions, these surface Ig molecules had a heavy chain that appeared slightly larger (approximately 81,000 daltons) than mu-chain (approximately 79,000 daltons), and light chains of approximately 25,000 daltons. Complete clearance of membrane-bound IgM reactive with an anti-mu allotype antiserum left similar molecules precipitate by monoclonal anti-light chain antibody. These non-IgM molecules could be detected on the surface of lymphocytes from blood, spleen, bursa and the B cell line RAV-1, but not from thymus or blood from an agammaglobulinemic chicken. After capping of B cell surface IgM with anti-mu, immunofluorescent staining with anti-light chain antibody revealed residual Ig molecules disturbed across the surface of more than 90% of the IgM-bearing cells. The data suggest the existence of an avian homologue of mammalian IgD. Affinity-purified goat anti-mu antibodies and a fourth monoclonal anti-mu antibody reacted with both IgM and the putative IgD molecules, which suggests that the IgD homologue shares at least one common determinant with chicken IgM.     

High level expression of functional human IgMs in human PER.C6® cells

Natural IgM antibodies play an important role in the body’s defense mechanisms against transformed cells in the human body and are currently being exploited both in prognoses of malignant lesions and in the therapy of cancer patients. However, despite growing interest and clinical promise, thus far the IgM class of antibodies has failed to gain widespread commercial interest as these are considered to be difficult to produce recombinantly. IgMs are polymeric and have a relatively large mass. In addition, IgM molecules are heavily glycosylated and, when produced in non-human cell lines, they may contain non-human glycan structures which may be potentially immunogenic. Clearly, production systems capable of expressing human recombinant IgM antibodies are needed. We have successfully used PER.C6® cells – a human cell line - to generate three separate human recombinant monoclonal IgMs in suspension cultures in protein-free medium. All three of the IgMs were constructed with joining (J) chain and were expressed in the pentameric form. One of the IgMs was also expressed as a hexamer without J chain. Clones with cell specific productivities greater than 20 pg/cell/day were generated, which led to yields of 0.5 g/L to 2g/L in fed-batch production. All the IgMs expressed were biologically active as shown in binding and cytotoxicity assays. These studies demonstrate the potential of PER.C6® cells for the production of high levels of functional recombinant IgM and other polymeric molecules, using a straightforward and rapid stable cell line generation method.     

B cell response pathways regulated by IL-5 and IL-2. Secretory microH chain-mRNA and J chain mRNA expression are separately controlled events

We have examined the effect of IL-5 and/or IL-2 on the expression of the secretory form of microH chain (microsecond) and J chain mRNA in a homogeneous neoplastic B cell clone, in which proliferation, IL-2R up-regulation and entry into the IgM secretory state are separately controlled events. The IL-5 signal triggers a partial induction of CL-3 cells into the IgM secretory state, characterized by a striking increase of microsecond mRNA expression and an increase in the ratio of the secretory to membrane forms of microH chain mRNA, with a modest increase of J chain mRNA. In contrast, amplification of J chain mRNA is accomplished by the late-acting B cell differentiation stimulus, IL-2, acting on IL-5-pretreated CL-3 cells or acting simultaneously with IL-5 on CL-3 cells. Such dually stimulated cells now are fully induced into IgM secreting cells. These results define the relative roles of IL-5 and IL-2 in B cell differentiation by showing important regulatory effects at the mRNA level. In addition, these results substantiate that appearance of mRNA for J chain, a molecule key to the formation of pentameric IgM, is a limiting factor for high level IgM secretion. The separate control of microsecond and J chain mRNA found in CL-3 cells stimulated with IL-5 and IL-2 elucidates a molecular mechanism by which these two lymphokines synergize in the development of CL-3 cells into IgM secreting cells.     

High level expression of functional human IgMs in human PER.C6? cells

Natural IgM antibodies play an important role in the body''s defense mechanisms against transformed cells in the human body and are currently being exploited both in prognoses of malignant lesions and in the therapy of cancer patients. However, despite growing interest and clinical promise, thus far the IgM class of antibodies has failed to gain widespread commercial interest as these are considered to be difficult to produce recombinantly. IgMs are polymeric and have a relatively large mass. In addition, IgM molecules are heavily glycosylated and, when produced in non-human cell lines, they may contain non-human glycan structures which may be potentially immunogenic. Clearly, production systems capable of expressing human recombinant IgM antibodies are needed. We have successfully used PER.C6® cells—a human cell line—to generate three separate human recombinant monoclonal IgMs in suspension cultures in protein-free medium. All three of the IgMs were constructed with joining (J) chain and were expressed in the pentameric form. One of the IgMs was also expressed as a hexamer without J chain. Clones with cell specific productivities greater than 20 pg/cell/day were generated, which led to yields of 0.5 g/L to 2g/L in fed-batch production. All the IgMs expressed were biologically active as shown in binding and cytotoxicity assays. These studies demonstrate the potential of PER.C6® cells for the production of high levels of functional recombinant IgM and other polymeric molecules, using a straightforward and rapid stable cell line generation method.Key words: PER.C6®, IgM, expression, hexamer, pentamer, J chain     

Activation of murine macrophage-like cells by granulocytin

Granulocytin, a C-type lectin from Sarcophaga peregrina (flesh fly), stimulated glucose consumption and cytokine production by the mouse macrophage-like cell line J774.1. When J774.1 cells were pretreated with tunicamycin, their granulocytin-dependent TNF-alpha production was greatly reduced. These results suggest that the stimulus of granulocytin is transmitted to J774.1 cells via the carbohydrate chain of granulocytin receptors located on their surface.     

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.     

The incorporation of J chain into reassembled human immunoglobulin M

Human IgM (immunoglobulin M) was reduced with 24mm-mercaptoethylamine. This atreatment resulted in complete dissociation to IgMs subunits and free J chain. Intr-subunit interchain disulphide bonds remained intact. The mixture then was encouraged to reoxidize. The schlieren pattern of the reoxidized mixture showed the presence of a considerable quantity of IgM in addition to residual IgMs. The isolated reassembled IgM did not dissociate in 5m-guanidinium hydrochloride. It apparently contained the same amount of covalently attached J chain as did native IgM. The J chain was a part of the high-molecular-weight Fc fragment obtained from the reassembled IgM.     

Rearrangements and deletions of immunoglobulin heavy chain genes in the double-producing B cell lymphoma I.29.

The B cell lymphoma I.29 consists of a mixture of cells expressing membrane-bound immunoglobulin M (IgM) (lambda) and IgA (lambda) of identical idiotypes. Whereas most of the cells express either IgM or IgA alone, 1 to 5% of the cells in this tumor express IgM and IgA simultaneously within the cytoplasm and on the cell membrane (R. Sitia et al., J. Immunol. 127:1388-1394, 1981; R. Sitia, unpublished data). When IgM+ cells are purified from the lymphoma and passaged in mice or cultured, a portion of the cells convert to IgA+. These properties suggest that some cells of the I.29 lymphoma may undergo immunoglobulin heavy chain switching, although it is also possible that the mixed population was derived by a prior switching event in a clone of cells. We performed Southern blotting experiments on genomic DNAs isolated from populations of I.29 cells containing variable proportions of IgM+ and IgA+ cells and on a number of cell lines derived from the lymphoma. The results were consistent with the deletion model for heavy chain switching, as the IgM+ cells contained rearranged mu genes and alpha genes in the germ line configuration on both the expressed and nonexpressed heavy chain chromosomes, whereas the IgA+ cells had deleted both mu genes and contained one rearranged and one germ line alpha gene. In addition, segments of DNA located within the intervening sequence 5' to the mu gene, near the site of switch recombination, were deleted from both the expressed and the nonexpressed chromosomes. Although mu genes were deleted from both chromosomes in the IgA+ cells, the sites of DNA recombination differed on the two chromosomes. On the expressed chromosome, Smu sequences were recombined with S alpha sequences, whereas on the nonexpressed chromosome, Smu sequences were recombined with S gamma 3 sequences.     

The control of IgM expression in a murine B cell lymphoma

The regulation of IgM expression was studied in clones derived from a murine B lymphocyte cell line, WEHI279.1. During normal B cell development IgM heavy chain synthesis increases concomitantly with heightened IgM secretion and reduced cell-surface IgM. However, in these subclones, the levels of membrane-bound and secreted IgM were regulated independently of one another. The amount of IgM secreted by the cells was tightly coupled to the amount of heavy chain synthesis, suggesting that the major control of secretion is pretranslational. Surface IgM exhibited a more complex regulation, with both pre- and posttranslational components. Variation in the expression of both forms of IgM occurred at high frequency. Although IgM expression follows a unidirectional pathway in nontransformed cells, the variability in these tumor cells was reversible and cellautonomous. High levels of phenotypic variability may be important in the ability of transformed cells to escape the immune response.     

Human serum IgM glycosylation: identification of glycoforms that can bind to mannan-binding lectin

The glycoprotein IgM is the major antibody produced in the primary immune response to antigens, circulating in the serum both as a pentamer and a hexamer. Pentameric IgM has a single J chain, which is absent in the hexamer. The mu (heavy) chain of IgM has five N-linked glycosylation sites. Asn-171, Asn-332, and Asn-395 are occupied by complex glycans, whereas Asn-402 and Asn-563 are occupied by oligomannose glycans. The glycosylation of human polyclonal IgM from serum has been analyzed. IgM was found to contain 23.4% oligomannose glycans GlcNAc2Man5-9, consistent with 100% occupancy of Asn-402 and 17% occupancy of the variably occupied site at Asn-563. Mannan-binding lectin (MBL) is a member of the collectin family of proteins, which bind to oligomannose and GlcNAc-terminating structures. A commercial affinity chromatography resin containing immobilized MBL has been reported to be useful for partial purification of mouse and also human IgM. Human IgM glycoforms that bind to immobilized MBL were isolated; these accounted for only 20% of total serum IgM. Compared with total serum IgM, the MBL-binding glycoforms contained 97% more GlcNAc-terminating structures and 8% more oligomannose structures. A glycosylated model of pentameric IgM was constructed, and from this model, it became evident that IgM has two distinct faces, only one of which can bind to antigen, as the J chain projects from the non-antigen-binding face. Antigen-bound IgM does not bind to MBL, as the target glycans appear to become inaccessible once IgM has bound antigen. Antigen-bound IgM pentamers therefore do not activate complement via the lectin pathway, but MBL might have a role in the clearance of aggregated IgM.     

Secondary V(D)J rearrangements and B cell receptor-mediated down-regulation of recombination activating gene-2 expression in a murine B cell line

It has recently become clear that recombination of Ig genes is not restricted to B cell precursors but that secondary rearrangements can also occur under certain conditions in phenotypically immature bone marrow and peripheral B cells. However, the nature of these cells and the regulation of secondary V(D)J recombination in response to B cell receptor (BCR) stimulation remain controversial. In the present study, we have analyzed secondary light chain gene rearrangements and recombination activating gene (RAG) expression in the surface IgM+, IgD- murine B cell line, 38C-13, which has previously been found to undergo kappa light chain replacement. We find that 38C-13 cells undergo spontaneous secondary Vkappa-Jkappa and RS rearrangements in culture, with recombination occurring on both productive and nonproductive alleles. Both 38C-13 cells and the Id-negative variants express the RAG genes, indicating that the presence of RAG does not depend on activation via the 38C-13 BCR. Moreover, BCR cross-linking in 38C-13 cells leads to a rapid and reversible down-regulation of RAG2 mRNA. Therefore, 38C-13 cells resemble peripheral IgM+, IgD- B cells undergoing light chain gene rearrangement and provide a possible in vitro model for studying peripheral V(D)J recombination.     

Molecular components of the B cell antigen receptor complex of class IgD differ partly from those of IgM

Two classes of immunoglobulin, IgM and IgD, are present as antigen receptors on the surface of mature B lymphocytes. We show here that IgD molecules are noncovalently associated in the B cell membrane with a heterodimer consisting of two proteins of 35 kd (IgD-alpha) and 39 kd (Ig-beta), respectively. The two novel proteins are not found in the IgD-expressing myeloma J558L delta m, which fails to bring IgD antigen receptor onto the cell surface. In a surface IgD positive variant line of this myeloma, however, membrane-bound IgD molecules are associated with the heterodimer, suggesting that the formation of an antigen receptor complex is required for surface IgD expression. We further demonstrate that the IgD-associated heterodimer differs partly from that of the IgM antigen receptor and that its binding to the heavy chain only requires the presence of the last constant domain and the transmembrane part of the delta m chain.     

Characterization of the immunodeficiency of RIIS/J [corrected] mice. I. Association with the CD5 (LY-1) [corrected] B cell lineage

RIIIS/J mice produce low antibody responses to several polysaccharide Ag of bacterial origin. They have low levels of serum IgM and IgG3 and high levels of serum IgG2a and IgG2b. Low serum IgM and IgG3 have been attributed to a low frequency of CD5 (Ly-1) B cells, which play an important role in the production of natural antibodies. Indeed, RIIIS/J mice have a low frequency of CD5 (Ly-1)+, IgM bright+, Ly-5 (B220)dull+ (i.e., CD5 (Ly-1) B) cells in their peritoneum. RIIIS/J mice treated with LPS produce a low anti-bromelain-treated mouse RBC splenic plaque-forming cell response and a normal anti-mouse transferrin splenic PFC response. Those data are compatible with the fact that CD5 (Ly-1) B cells contain the precursors of B lymphocytes secreting anti-bromelain-treated mouse RBC antibody. However, they have a higher frequency of IgM bright+, Mac-1+ cells in their peritoneum. These cells represent the CD5 (Ly-1) "sister population" of CD5 (Ly-1) B cells described by others. This suggests that characteristics usually associated with the CD5 (Ly-1) lineage are applicable only to the CD5 (Ly-1)+ Mac-1+ IgM+ population, but not the related CD5 (Ly-1)- Mac-1+ IgM+ population. RIIIS/J mice should thus prove a valuable model to study the CD5 (Ly-1) B cell lineage.     

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.     

Selective proteolysis of the J chain component in human polymeric immunoglobulin.

To clarify the losses that have been observed in the J chain portion of human IgM and IgA, were carried out studies on the enzymatic susceptibility of the J polypeptide. When Waldenstr?m macroglobulins and myeloma IgA polymers were subjected to limited proteolysis with various endopeptidases, only subtilisin was found to attack the J chain component. The pattern of cleavage was a function of the polymer species. The J chain in IgM was highly susceptible to digestion, quantitative cleavage being achieved at very low enzyme to IgM ratios and without significant changes in the remaining pentamer structure. Analyses of the digestion products showed that the initial cleavage occurred at an exposed region midway in the J sequence and was followed by extensive degradation of the carboxy-terminal segment. These findings indicated that the observed loss of the IgM J component can be explained by the inadvertent introduction of subtilisin in vitro or by the attack of in vivo enzymes with a specificity similar to subtilisin. In contrast, the IgA J chain was found to be much more resistant to subtilisin proteolysis; its cleavage required higher enzyme concentrations and was accompanied by significant degradation of the alpha-chains. Thus, it appears unlikely that the IgA J polypeptide is degraded by either in vitro or in vivo enzymes unless its accessibility is first enhanced by changes in the IgA Fc structure.