Cell membrane IgD: demonstration of IgD on human lymphocytes by enzyme-catalyzed iodination and comparison with cell surface Ig of mouse, guinea pig, and rabbit.

A substantial fraction of human cord blood and peripheral blood lymphocytes have recently been shown to bear IgD. Although IgD has not been identified in mice, it has been suggested that it is also a major surface immunoglobulin of murine lymphocytes. Thus, lactoperoxidase-catalyzed iodination of surface immunoglobulin of mouse spleen and lymph node cells reveals the existence of an IgH chain differing from mu, gamma, and alpha-chain both antigenically and by mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This new H chain class has been previously proposed to be the mouse homologue of delta-chain. In this paper, we analyzed human, mouse, guinea pig, and rabbit lymphoid cell membrane Ig by lactoperoxidase-catalyzed iodination, extraction with non-ionic detergent precipitation with a variety of specific anti-Ig sera, and electrophoresis of dissolved reduced precipitates on sodium dodecyl sulfate-polyacrylamide gels. Our studies confirm the previous reports of a new mouse cell membrane H chain with a mobility more rapid than that of mu-chain. However, we fail to detect a molecule with this electrophoretic mobility on the surface of guinea pig or rabbit lymph node and spleen cells. Moreover, neither anti-kappa nor anti-delta antibody precipitates a molecule with an H chain of this mobility from labeled extracts of human cord blood or peripheral blood lymphocytes. Cell surface delta was identified on both human cord blood and peripheral blood lymphocytes, but it proved to have mobility similar to human and mouse mu-chain. This result indicates either that mouse delta-chain has an electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels which differs appreciably from that of human membrane delta-chain or that the newly described mouse H chain is not the homologue of human delta-chain.     

Regulation of endoplasmic reticulum stress proteins in COS cells transfected with immunoglobulin mu heavy chain cDNA

The mechanism by which endoplasmic reticulum (ER) stress proteins are induced by the accumulation of incompletely assembled or malfolded proteins in the ER is poorly understood. The 78-kDa glucose-regulated protein (BiP), one of the ER stress proteins, has often been detected in stable complexes with these accumulated proteins. We have transfected COS cells with an immunoglobulin (Ig) mu heavy chain expression plasmid. Expressed mu-chain accumulated in the cells and formed stable complexes with BiP. As a result, the synthesis of three ER stress proteins, BiP, the 94-kDa glucose-regulated protein (GRP94/ERp99), and ERp72, was increased as were their mRNA levels. In addition, the degradation rate of BiP was increased, possibly because of its interaction with mu-chain. Cotransfection of the mu-chain plasmid with an Ig lambda light chain expression plasmid resulted in the appearance of mu-chain in the media in a covalent complex with lambda-chain. An intracellular consequence of this was a reduction in the levels of BiP.mu-chain complex, and a diminished stimulation in the synthesis of the ER stress proteins. These results suggest that the BiP.mu-chain complex in the ER may be involved in the signaling pathway for the induction of ER stress proteins and may represent one regulatory mechanism operating in differentiating B-lymphocytes.     

The DJH complex remains active in recombination to VH segments after the loss of mu-chain expression in mu-positive pre-B cells

AT11-2 is an Abelson virus-transformed B precursor cell line which is capable of differentiating Ig- from mu+ cells via functional recombination of VH segments to preexisting DJH complexes. We describe here that after a mu+ subclone (VDJ+/DJ) generated from Ig- AT11-2 (DJ/DJ) cells by in vitro functional VH to DJH recombination subsequently lost mu-chain expression either by the recombination of a pseudo VH segment to the VHDJH+ allele or by the deletion of VHDJH+ allele, a novel productive joining of VH segments to the preexisting DJH complex occurred. These results indicated that VH to VHDJH rearrangement was not suppressed in mu-chain producing cells and that the DJH complexes still remained active in the recombination to VH segments after the loss of mu-chain expression. Our results may also suggest that VH to DJH rearrangement, but not VH to VHDJH rearrangement, is suppressed in mu-chain producing cells to maintain allelic exclusion. Our cell differentiation system should continue to be valuable for elucidating the mechanism of suppression and associated implications regarding allelic exclusion.     

Ocular herpes simplex virus infection is diminished by depletion of B lymphocytes

Rabbit anti-mouse mu-chain serum was used to deplete specifically IgM-bearing B cells and B cell function in BALB/c mice. B cell-depleted and normal littermates were infected via the cornea with herpes simplex virus type 1 (HSV-1) at 4 to 5 wk of age. B cell-depleted mice had a reduced number of lymphocytes bearing surface immunoglobulin and a greatly reduced ability to produce antibody. Mortality was reduced from 89% in normal controls to 42% in B cell-depleted mice (p less than 0.001) and peak virus shedding from eyes was also reduced as much as 90% in the B cell-depleted mice (p less than or equal to 0.0005). These results support the hypothesis that a B cell function of normal mice somehow contributes directly or indirectly to the enhanced mortality of mice infected with HSV.     

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.     

Antibody conjugates mimic specific B cell presentation of antigen: relationship between T and B cell specificity

We developed antibody conjugates by covalently coupling antibodies against mouse mu-chain and monoclonal antibodies against nominal antigen, myoglobin, as a tool for antigen presentation and as a model of specific presentation of antigen by antigen-specific B cells and T-B interaction. In the presence of the antibody conjugates, myoglobin-specific Iad-restricted cloned T cells proliferated at 1000-fold lower concentration of myoglobin than the stimulatory concentration without the conjugates. This enhanced presentation was observed only when Iad spleen cells were 1000 R-irradiated but not 3300 R-irradiated, consistent with B cell presentation. The simple mixture of each component of the conjugates had no enhancement effects. The conjugates per se had no mitogenic effects on either splenic B cells or the cloned T cells at concentrations employed for antigen presentation. The conjugates reduced the number of antigen-presenting cells required for the maximal response but did not change the kinetics of response. The enhanced presentation by the conjugates required a genetically restricted interaction with B cells. Antigen specificity of the enhanced presentation was confirmed by using various T cell clones or lines with different antigen specificities and different conjugates constructed with monoclonal antibodies of known epitope specificity. The enhanced presentation was significantly inhibited by competition with exogenous mouse IgM or anti-mouse mu-chain but was not significantly inhibited by monoclonal antibodies against Fc receptor. Thus, conjugate-coated B cells serve as models for myoglobin-specific B cells in that they can take up specific antigens at extremely low concentration and can present the antigen to specific T cells. This model system can be applied to any antigen and any species without the need to develop antigen-specific B cell clones, which is not yet possible for most antigens and species of experimental animals. This system allowed us to investigate the relationship between T cell epitope and B cell epitope when these cells interact with each other in an antigen-specific and Ia-restricted manner. Experiments using antibody conjugates of different monoclonal antibodies against myoglobin and various myoglobin-specific cloned T cells of known antigen specificity revealed that there are some particular combinations in which much more limited enhancement of antigen presentation is observed.(ABSTRACT TRUNCATED AT 400 WORDS)     

Differentiation of growth signal requirement of B lymphocyte precursor is directed by expression of immunoglobulin.

During B cell differentiation, at least three stages can be defined in terms of their growth signal requirement by using two different growth signals, which are recombinant interleukin 7 (IL-7) and a stromal cell clone PA6 which does not produce IL-7; first a PA6 dependent stage, second a PA6 + IL-7 dependent stage and third an IL-7 dependent stage. In order to test the possibility that this differentiation of growth signal requirement is controlled by the expression of functional immunoglobulin molecules, we have investigated the frequencies of PA6 + IL-7 dependent and IL-7 dependent cells which are present in the bone marrow of either mu-chain or kappa-chain gene transgenic mice. In a mu-chain gene transgenic mouse, the frequency of PA6 + IL-7 dependent cells is selectively reduced, while that of IL-7 dependent cells is selectively reduced in a kappa-chain gene transgenic mouse. This result suggests that expression of a functional mu-chain gene drives PA6 + IL-7 dependent cells to differentiate into the subsequent IL-7 dependent stage. Likewise, when mu-chain positive IL-7 dependent cells express a functional light-chain gene, their growth signal requirement changes into an IL-7 unreactive stage.     

Immunoregulatory factors derived from human tumors. II. Partial purification and further immunobiochemical characterization of a human sarcoma-derived immunosuppressive factor expressing HLA-DR and immunoglobulin-related determinants

An immunoregulatory factor (IRF) that suppresses Con A-mediated peripheral blood mononuclear cell (PBM) proliferative responses was partially purified by DEAE anion exchange chromatography and affinity chromatography from a 3 M KCl extract of a human liposarcoma. The factor (m.w. = 70K) co-purified with albumin, monitored by two-dimensional gel electrophoresis, and demonstrated a heterogeneous isoelectric point (pI 7.6-7.8). Xenoantisera produced against the DEAE-purified fraction and coupled to Affigel 10 removed suppressive activity that could subsequently be eluted by glycine-HCl, pH 3.5. An anti-albumin column partially removed activity, but the unbound 70K factor could still be detected in the column effluent. A xenoantiserum to this 70K effluent coupled to acrylamide beads completely removed the immunosuppressive activity in immunodepletion experiments. Further direct binding enzyme-linked immunoassays (ELISA) and solid-phase immunoabsorption experiments with monoclonal antibodies to human anti-HLA-DR framework determinants and a constant region of the IgM mu-chain demonstrated determinants on the 70K factor recognized by these antibodies.     

Oligosaccharide processing at individual glycosylation sites on MOPC 104E immunoglobulin M. Differences in alpha 1,2-linked mannose processing

Processing of the asparagine-linked oligosaccharides at the known glycosylation sites on the mu-chain of IgM secreted by MOPC 104E murine plasmacytoma cells was investigated. Oligosaccharides present on intracellular mu-chain precursors were of the high mannose type, remaining susceptible to endo-beta-N-acetylglucosaminidase H. However, only 26% of the radioactivity was released from [3H]mannose-labeled secreted IgM glycopeptides, consistent with the presence of high mannose-type and complex-type oligosaccharides on the mature mu-chain. [3H]Mannose-labeled cyanogen bromide glycopeptides derived from mu-chains of secreted IgM were isolated and analyzed to identify the glycopeptide containing the high mannose-type oligosaccharide from those containing complex-type structures. [3H]Mannose-labeled intracellular mu-chain cyanogen bromide glycopeptides corresponding to those from secreted IgM were isolated also, and the time courses of oligosaccharide processing at the individual glycosylation sites were determined. The major oligosaccharides on all intracellular mu-chain glycopeptides after 20 min of pulse labeling with [3H]mannose were identified as Man8GlcNAc2, Man9GlcNAc2, and Glc1Man9GlcNAc2. Processing of the oligosaccharide destined to become the high mannose-type structure on the mature protein was rapid. After 30 min of chase incubation the predominant structures of this oligosaccharide were Man5GlcNAc2 and Man6GlcNAc2 which were also identified on the high mannose-type oligosaccharide of the secreted mu-chain. In contrast, processing of oligosaccharides destined to become complex type was considerably slower. Even after 180 min of chase incubation, Man7GlcNAc2 and Man8GlcNAc2 were the predominant structures at some of these glycosylation sites. The isomeric structures of Man8GlcNAc2 obtained from all of the glycosylation sites were identical. Thus, the different rates of processing were not the result of a different sequence of alpha 1,2-mannose removal.     

Increased avidity of mutant IgM antibodies caused by the absence of COOH-terminal glycosylation of the mu H chain.

We have previously described the isolation of two hybridoma variants secreting higher avidity IgM (D5 and 7F5), starting from the E11 hybridoma cell line, which produces an antibody specific for the A Ag of the ABO blood group system. In order to explain at the molecular level this increased reactivity, cDNA encoding the H and L chains of the E11, D5, and 7F5 mAb were cloned and sequenced. Comparison of the nucleotide sequences showed a single point mutation in each of the two mAb produced by the hybridoma variants. The mutations were both located in the H chain C region and caused a Ser to Phe substitution at position 565 in the D5 mAb and a Asn to Tyr substitution at position 563 in the 7F5 mAb. Both substitutions modified the consensus glycosylation sequence (Asn-X-Ser/Thr) located in the tail piece of the secretory mu-chain. The absence of glycosylation at this site was confirmed by CNBr cleavage of the [14C]mannose-labeled mAb. The two single point mutations were solely responsible for the increased avidity of the antibodies, as confirmed by site-directed mutagenesis of the E11 mu-chain and serologic analysis of the mutated E11 antibodies. We conclude that the absence of glycosylation at Asn 563 is responsible for the increased avidity of the mutant, possibly by altering the quaternary structure of the IgM polymer. To our knowledge, this is the first report that point mutations in the H chain C region can influence the reactivity of IgM mAb.     

The effect of peptide deletions on the glycosylation of murine immunoglobulin M heavy chains

The glycosylation and processing of the asparagine-linked oligosaccharides at individual glycosylation sites on the mu-chain of murine immunoglobulin M were investigated using variant cell lines that synthesize and secrete IgM heavy chains with known peptide deletions. Normal murine IgM has five N-linked oligosaccharides in the constant region of each heavy or mu-chain. Each mu-chain has four complex-type oligosaccharides as well as a single high mannose-type oligosaccharide near the carboxyl terminus of the molecule. The peptide deletion of the C mu 1 constant region domain in the heavy chains synthesized by one variant cell line did not prevent subsequent glycosylation at more distal glycosylation sites. In fact, the presence of this deletion resulted in more complete glycosylation at the C-terminal glycosylation site. Evaluation of glycopeptides containing individual glycosylation sites by Concanavalin A-Sepharose indicated that this deletion had no significant effect on the processing of structures from high mannose-type to complex-type oligosaccharide chains. In contrast, a deletion of the C-terminal peptide region of the heavy chain of IgM synthesized by a second variant cell line resulted in intracellular processing to more highly branched oligosaccharide structures at several of the glycosylation sites not involved in the deletion.     

The IgM-associated protein mb-1 as a marker of normal and neoplastic B cells.

Recent evidence indicates that the transmembrane form of IgM on murine and human B lymphocytes is physically associated with at least two proteins, forming a disulfide-linked dimer, which may control cell surface expression of IgM and also play a role in signal transduction after Ag binding (by analogy with the TCR-associated CD3 components in T lymphocytes). We have used mAb and polyclonal antibodies against an intracytoplasmic epitope on one of these polypeptides (previously identified in murine B cells as the product of the B cell specific mb-1 gene) to study the distribution of the IgM-associated dimer in human cells. By immunocytochemical staining of normal and neoplastic B cells, we show that the human mb-1 protein appears early in B cell differentiation, probably before expression of cytoplasmic mu-chain, and persists until the plasma cell stage, where it is seen as an intracytoplasmic component. According to immunohistologic analysis of reactive lymphoid tissue and lymphoma samples, mb-1 protein is completely B cell specific. Anti-mb-1 also labels B cell areas in tissues from seven different mammalian species. Finally, the Ig-associated dimer could be isolated from human hairy-cell leukemia cells in high purity and yield by affinity chromatography using anti-mb-1 antibody. Mice immunized with this material have produced a strong polyclonal response, so that it should now be possible to prepare a panel of new mAb reactive with different epitopes on both mb-1 and on its associated polypeptide(s).     

Non-viral cellular substrates for human immunodeficiency virus type 1 protease

A computer search revealed 10 proteins with homology to the sequence we originally identified in vimentin as the site of cleavage by human immunodeficiency virus type 1 (HIV-1) protease. Of these 10 proteins (actin, alpha-actinin, spectrin, tropomyosins, vinculin, dystrophin, MAP-2, villin, TRK-1 and Ig mu-chain), we show that 4 of the first 5 were cleaved in vitro by this protease, as are MAP-1 and -2 [(1990) J. Gen. Virol. 71, 1985-1991]. In these proteins, cleavage is not restricted to a single motif, but occurs at many sites. However, cleavage is not random, since 9 other proteins including the cytoskeletal proteins filamin and band 4.1 are not cleaved in the in vitro assay. Thus, the ability of HIV-1 protease to cleave specific components of the cytoskeleton may be an important, although as yet unevaluated aspect of the life cycle of this retrovirus and/or may directly contribute to the pathogenesis observed during infection.     

Activation of cation transport by lymphokines in B cells without induction of DNA synthesis or immunoglobulin gene transcription

We report on an experimental model that permitted us to evaluate the biologic relevance of membrane-associated biochemical events with respect to cell proliferation and maturation, each induced by distinct sets of signals. Antigen-affinity-enriched murine B cells cultured in the presence of a proliferative signal induced by LPS showed activation of Na+/K+ ATPase and enhanced the uptake of proline, followed by RNA, protein, and DNA synthesis, without the generation of antibody. Stimulation with both the proliferative signal(s) and the maturation signal(s) derived from lymphokines of an EL-4 thymoma induced B cells to proliferate and synthesize mRNA encoding mu-chain of IgM and to mature into IgM-secreting cells. Most important, the secretory product of EL-4, in the absence of LPS, activated Na+/K+ ATPase but failed to stimulate uptake of proline and synthesis of DNA or mu-specific mRNA. A similar response was observed in splenocytes depleted of T cells and in unfractionated spleen cells. Thus a component secreted by EL-4 can induce some of the early molecular events characteristic of the proliferative response but lacks the ability to initiate blast transformation and DNA synthesis.     

A region of the immunoglobulin-mu heavy chain necessary for forming pentameric IgM

In order to define the molecular requirements for IgM pentamer formation, we have isolated several mutant hybridomas which produce predominantly monomeric IgM. For one such mutant, 102, we synthesized a cDNA clone of its mu-mRNA, and found an in-frame 39-bp deletion, which thus encodes a mu-chain lacking amino acids 550-562, a region spanning the fourth constant domain and the tail of the mu-chain. To prove that this deletion is sufficient to block pentamer formation, we used site-directed mutagenesis to construct a mu-gene lacking these 39 bp, and have shown that the expression of this altered mu-gene results in the production of monomeric IgM.     

Accessibility of tryptophan residues in immunoglobulin M molecule as an indicator of its conformational variability

The accessibility of tryptophan residues in immunoglobulin M to modification with the Koshland reagent (2-hydroxy-5-nitrobenzyl bromide) was used as an indicator of its conformational variability. Of 14 tryptophan residues (per HL-fragment) in the native IgM, only one (presumably Trp312 in the mu-chain) was the most accessible. Irreversible acid- or temperature-induced conformational changes of IgM increased almost 2-fold the number of accessible tryptophan residues. After partial enzymatic deglycosylation of IgM (especially by an intense splitting of mannose), all tryptophan residues became inaccessible. Modification of the most accessible tryptophan residue increased 2- to 3-fold the number of tyrosine residues accessible to nitration with tetranitromethane. Using the spin label method, it was demonstrated that modification of four tryptophan residues in IgM considerably decreased the mobility of the Cmu 3 domain together with an essential drop in. the solubility of the modified IgM.     

Splenic B cells from CBA/N mice acquire responsiveness to anti-immunoglobulin after a brief treatment with pronase

Although splenic B cells of CBA/N mice do not synthesize DNA in response to anti-mouse IgM (mu-chain specific), the cells respond readily to Sepharose linked anti-mu. Subsequent to a brief treatment with pronase, CBA/N splenocytes exhibited anti-mu-mediated DNA synthesis at 40 to 100% of the DNA synthetic capacity detected with Sepharose linked anti-mu. Furthermore, spleen cell populations treated with anti-Thy-1.2 and complement or populations purified on anti-immunoglobulin-coated Petri plates (greater than 90% surface immunoglobulin positive) acquired responsiveness to anti-mu after pronase treatment.