Microenvironmental studies of pre-B and B cell development in human and mouse fetuses

Immunofluorescence techniques were used to trace the development of cells expressing mu heavy chains in humans and mice. IgM B cells were distinguished from pre-B cells by their additional expression of kappa or lambda light chains. Generation of pre-B and progeny B cells was evident in hemopoietic fetal liver and bone marrow, but not in thymus, heart, lung, spleen, kidney, and placental tissues. Pre-B and B cells, in a ratio of 2 to 1, were abundant in sections of hemopoietic liver and in bone marrow from 12- to 15-wk-old human fetuses, whereas these cells were rare in nonhemopoietic liver samples obtained beyond the 34th week. In mouse fetal liver mu+ cells appeared first around the 12th day of gestation and increased in frequency throughout the third trimester. On day 17 of gestation, kappa light chain expression by 1% of mu+ cells was noted, and the percentage of kappa+/mu+ cells increased progressively to more than 80% by 5 days after birth. Pre-B and B cells were interspersed among myeloid and more abundant erythropoietic cellular elements in the extrasinusoidal areas adjacent to hepatic cords. A loose clustering or "starburst" distribution pattern of pre-B cells became evident around day 17. These observations suggest a model for in situ generation of pre-B and progeny B cells in the hemopoietic fetal liver. In the midst of more numerous erythropoietic elements, immunoglobulin-negative precursors divide to generate a loose colony of mu+ pre-B cells that divide again before giving rise to a wave of IgM B cells.     

Immunoglobulin light chains dictate vesicular transport-dependent and -independent routes for IgM degradation by the ubiquitin-proteasome pathway

Degradation of IgM mu heavy chains in light chain-negative pre-B cells is independent of vesicular transport, as is evident by its insensitivity to brefeldin A or cell permeabilization. Conversely, by the same criteria, degradation of the secretory mu heavy chain in light chain-expressing B cells depends on vesicular transport. To investigate whether the presence of conventional light chains or the developmental stage of the B-lymphocytes dictates the degradative route taken by mu, we express in 70Z/3 pre-B cells either lambda ectopically or kappa by lipopolysaccharides-stimulated differentiation into B cells and show their assembly with mu heavy chains. The resulting sensitivity of mu degradation to brefeldin A and cell permeabilization demonstrates that conventional light chains, a hallmark of B cell differentiation, are necessary and sufficient to divert mu from a vesicular transport-independent to a vesicular transport-dependent degradative route. Although both routes converge at the ubiquitin-proteasome degradation pathway, only in light chain-expressing cells is vesicular transport a prerequisite for mu ubiquitination.     

Immunoglobulin synthesis and gene rearrangements in lymphoid cells transformed by replication-competent Rauscher murine leukemia virus: transformation of B cells at various stages of differentiation

Lymphoid cells transformed by Rauscher murine leukemia virus (R-MuLV) belonged to the B cell lineages. One group of cells exhibited Fc receptors but completely lacked immunoglobulin mu heavy and kappa light chains. The majority of the cells resemble pre-B type. They displayed mu chains but kappa chains were completely absent. Very rarely certain cells synthesized both mu and kappa chains. Based on the presence of Fc receptors and IgM synthesis the cells transformed by R-MuLV belonged to three B cell developmental stages. These cells were tested for immunoglobulin gene rearrangements using JH and CK probes. DNA from cell lines without any detectable levels of IgM mu exhibited embryonic as well as rearranged JH genes, whereas cells expressing IgM possess, in addition, productive and non-productive light chain gene rearrangements. The most terminally differentiated cell possesses JH and CK rearrangement associated with the synthesis of mu and kappa chains. Presumably the cells with rearranged JH and CK genes without immunoglobulin synthesis represent a developmental transition. We conclude that cells transformed by R-MuLV belonged to five step-wise compartments of B cell development. Our findings implicate definite sequential events of immunoglobulin gene rearrangement and expression during B cell development.     

Surface mu heavy chain expressed on pre-B lymphomas transduces Ca2+ signals but fails to cause growth arrest of pre-B lymphomas.

Little is known about the role of signals transduced by cell surface IgM (sIgM) expressed during early B cell development. A subclone (1.6) of the late pre-B cell lymphoma 70Z/3.12 was used to study signal transduction by surface mu heavy (H) chain before and after transition to the early immature B cell stage, and the functional consequences thereof. Although kappa L chain expression can be induced on 1.6 cells by LPS or cytokines, immunoprecipitations indicated that the non-induced 1.6 cells expressed mu H chain with an alternative protein(s) which may be a surrogate light chain(s). Consistent with this, anti-mu but not anti-kappa or anti-lambda antibodies caused transient Ca2+ mobilization in noninduced 1.6 cells. The Ca2+ signal was derived from both intracellular stores and Ca2+ influx in either noninduced cells or in cells that had been preinduced to express kappa L chain. Thus, the ability of mu H chain to mobilize Ca2+ as a second messenger does not depend upon the expression of mature L chains. The immature B lymphomas, WEHI-231 and CH1, express mature forms of IgM and undergo growth arrest when stimulated by anti-mu antibody. In contrast, signals generated by mu H chain on either noninduced or preinduced 1.6 cells or in the sIgM+ pre-B cell transfectant 300-19 mu lambda 36/8 did not cause growth arrest. These results suggest that mu H chain expressed on pre-B cells is capable of mobilizing Ca2+, but that this signal alone is insufficient to induce growth arrest in the pre-B cell.     

The immunoglobulin mu chains of membrane-bound and secreted IgM molecules differ in their C-terminal segments

The B lymphocytes synthesizes two forms of IgM molecules during its development from a stem cell to a mature antibody-secreting plasma cell. The monomeric receptor IgM molecule is affixed to the plasma membrane and triggers the later stages of B cell differentiation, whereas the pentameric secreted IgM molecule is an effector of humoral immunity. The structural differences between membrane-bound and secreted IgM molecules are reflected in the differences between their heavy or mu chains. We have previously determined the complete amino acid sequence of a murine secreted mu (microsecond) chain. In this study, we have compared the structures of the secreted and membrane-bound mu (micron) heavy chains by peptide mapping, micro-sequence and carboxypeptidase analyses. These studies demonstrate that the micron and microsecond chains are very similar throughout their VH, C mu 1, C mu 2, C mu 3 and C mu 4 domains. The micron and microsecond chains differ in the amino acid sequence of their C-terminal segments. These studies in conjunction with those carried out on the micron and microsecond mRNAs and the C mu gene suggest that the micron and microsecond chains from a given B cell are identical except for their 41 and 20 residue C-terminal segments, respectively. The amino acid sequence of the 41 residue C membrane terminal segment predicted from the corresponding micron mRNA is in agreement with all the protein studies reported in this paper.     

Posttranslational association of immunoglobulin heavy chain binding protein with nascent heavy chains in nonsecreting and secreting hybridomas

A rat monoclonal antibody specific for immunoglobulin (Ig) heavy chain binding protein (BiP) has allowed the examination of the association of BiP with assembling Ig precursors in mouse B lymphocyte-derived cell lines. The anti-BiP monoclonal antibody immunoprecipitates BiP along with noncovalently associated Ig heavy chains. BiP is a component of the endoplasmic reticulum and binds free intracellular heavy chains in nonsecreting pre-B (mu+, L-) cell lines or incompletely assembled Ig precursors in (H+, L+) secreting hybridomas and myelomas. In the absence of light chain synthesis, heavy chains remain associated with BiP and are not secreted. The association of BiP with assembling Ig molecules in secreting hybridomas is transient and is restricted to the incompletely assembled molecules which are found in the endoplasmic reticulum. BiP loses affinity and disassociates with Ig molecules when polymerization with light chain is complete. We propose that the association of BiP with Ig heavy chain precursors is a novel posttranslational processing event occurring in the endoplasmic reticulum. The Ig heavy chains associated with BiP are not efficiently transported from the endoplasmic reticulum to the Golgi apparatus. Therefore, BiP may prevent the premature escape and eventual secretion of incompletely assembled Ig molecules.     

Binding of free immunoglobulin light chains to VpreB3 inhibits their maturation and secretion in chicken B cells

The VpreB3 gene product was first characterized as an immunoglobulin (Ig) mu heavy chain-binding protein in mouse precursor B (pre-B) cells. Although its function is unknown, it has been proposed to participate in the assembly and transport of the pre-B cell receptor. We have identified a VpreB3 orthologous gene in chicken that is located close to the immunoglobulin light chain (LC) gene cluster and specifically expressed in the bursa of Fabricius. By overexpressing VpreB3 in the DT40 IgM(+) immature chicken B cell line, we have characterized VpreB3 as an endoplasmic reticulum-resident glycoprotein that binds preferentially to free IgLC. However, binding to IgHC is observed in IgLC-deficient DT40 cells. Interaction of VpreB3 with free IgLC is partly covalent and induces retention of free IgLC in the endoplasmic reticulum, preventing their secretion without affecting IgM surface expression. Our results demonstrate that this evolutionarily conserved molecule may play a role in the regulation of the maturation and secretion of free IgLC in B cells. We discuss possible implications in the regulation of the immune response.     

Two types of mu chain complexes are expressed during differentiation from pre-B to mature B cells.

Immunoglobulin mu chains synthesized in murine pre-B cells are known to be associated with surrogate light chains designated as omega (omega), iota (iota) and B34. In addition to these molecules, we identified the complexes of polypeptides (50, 40, 27 and 15.5 kd) associated with surface or intracellular mu chains of pre-B cell lines. Most of these polypeptides were continuously synthesized and associated with mu chains in virgin B cells lines, although some of them scarcely bound to the mu kappa dimer or mu 2 kappa 2 tetramer concomitantly present in the same clone or population. However, in mature B cells they were no longer detectable except B34. Cross-linking of micron chains on the surface of pre-B cells resulted in an increase in intracellular free Ca2+, indicating that the micron chain complex on the surface of pre-B cell lines acted as a signal transduction molecule. However, the receptor cross-linkage of pre-B cell lines did not induce the increased inositol phospholipid metabolism usually observed in virgin and mature B cell lines. These results suggest that, during the differentiation from pre-B to mature B cells, the cells express two types of mu chain complexes which exhibit different structures as a whole and possess different signal transducing capacities.     

A novel lymphocyte function-associated antigen (LFA-1): cellular distribution, quantitative expression, and structure

The derivation of a new human pre-B cell line from the leukocytes of a girl in the leukemic phase of lymphoblastic lymphoma is reported. Cells of this line, termed SMS-SB, synthesized mu but not light chains. These mu-chains were found only in the cytoplasm and were not secreted. SMS-SB cells bore HLA-DR determinants. They did not express the common ALL antigen or terminal deoxynucleotidyl transferase and, therefore, differed from previously described human pre-B leukemia cell lines. SMS-SB cells resembled certain Abelson leukemia virus-transformed mouse bone marrow cells. Except for the lack of formed mouse bone marrow cells. Except for the lack of mu secretion, the phenotype of SMS-SB cells was the same as the major subpopulation of marrow pre-B cells.     

Activation of V kappa gene rearrangement in pre-B cells follows the expression of membrane-bound immunoglobulin heavy chains.

During B cell development V kappa gene rearrangement seems to occur only in mu-positive pre-B cells. To study the role of the mu chain in the activation of the Ig kappa locus, we introduced expression vectors carrying different forms of the mu gene into null pre-B cells. The activation of the Ig kappa locus followed the expression of the membrane form (micron) of the mu chain. The expression of the secreted form (microS) did not result in the activation of the Ig kappa locus. We further show that both forms of the mu chain differ in their intracellular transport in pre-B cells.     

Regulation of membrane IgM expression in secretory B cells: translational and post-translational events.

IgM secreting cells express little or no membrane IgM. This is not always due to absence of the relevant mRNA. To investigate the synthesis and processing of membrane (micron) and secreted (microseconds) polypeptides in secretory B cells, myeloma cells were transfected either with a plasmid containing an intact mu gene or with one only capable of directing micron (not microseconds) mRNA synthesis. Although myeloma transfectants could make abundant levels of micron mRNA, they did not express IgM on the cell surface. In the myeloma host, micron mRNA is translated some 5-fold less efficiently than microseconds mRNA. However, this translational control does not totally preclude micron synthesis, indicating post-translational regulatory events. No difference between micron and microseconds chains could be detected in their rate of assembly with light chains or in their stability, although both types of heavy chain were degraded more rapidly when synthesized in the absence of light chain, or when the hydrophobic nature of the leader sequence was destroyed by site-directed mutagenesis. However, whereas intracellular microseconds chains in IgM-secreting plasmacytoma were found to be concentrated in the Golgi, the micron chains were mainly located in the endoplasmic reticulum. Retention in the endoplasmic reticulum is also observed for both micron and microseconds when synthesized in the absence of light chain. We propose that it is the expansion of the endoplasmic reticulum that accompanies B cell to plasma cell differentiation which is in part responsible for the down-regulation of surface IgM expression. Such a mechanism may also affect the expression of other surface proteins.     

B lymphocyte-associated antigens on terminal deoxynucleotidyl transferase-positive cells and pre-B cells in bone marrow of the rat

To investigate early stages of B lymphocytopoiesis in rat bone marrow (BM) before the expression of surface IgM (s mu), the populations of cytoplasmic mu-chain-positive (c mu+) pre-B cells and terminal deoxynucleotidyl transferase-positive (TdT+) cells were studied by double immunofluorescence microscopy. B lymphocytes that were s mu+ constituted 5%, c mu+s mu- pre-B cells 23%, and TdT+ cells 4% of nucleated cells in the BM of juvenile rats. TdT+ and pre-B cells ranged between 7 and 17 microns in diameter. TdT+ cells were slightly larger, with a modal diameter of 10.5 microns against 9 microns for pre-B cells. mu-Chains were absent from nearly all TdT+ cells. Their surface antigenic phenotype was studied by using a panel of mouse monoclonal antibodies (MAb) to rat B lymphocyte-associated antigens (Ig, Ia, and others) and T lymphocyte-associated antigens. Both pre-B cells and TdT+ lacked surface Ig and Ia but carried most of the other B lymphocyte-associated antigens analyzed. TdT+ and pre-B cells lacked those antigens found only on the T lineage. By using MAb HIS24 (detecting a non-Ig/Ia B lymphocyte-associated antigen) and fluorescence-activated cell sorting, TdT+ and pre-B cells were highly enriched. The results show that most TdT+ cells in rat BM are mu- but demonstrate strong similarity with pre-B cells in surface antigenic phenotype. Therefore, as suggested for man, a major proportion of rat BM TdT+ cells may be B lineage-cells before mu heavy chain gene expression.     

Regulation of early precursor B cell proliferation in mouse bone marrow: stimulation by exogenous agents mediated by macrophages in the spleen.

An increase in pre-B cell proliferation and B lymphocyte production in mouse bone marrow has previously been shown to follow the administration of various foreign agents in vivo. The responses of early precursor B cells before the expression of mu chains (pro-B cells) have now been examined, using double immunofluorescence labeling for terminal deoxynucleotidyl transferase (TdT) and B220 glycoprotein as detected by monoclonal antibody 14.8. A single injection of sheep red blood cells (SRBC) was followed by an increase in the number of cells in three defined populations of early precursor B cells lacking mu chains (TdT+ 14.8- cells, TdT+ 14.8+ cells, and 14.8+ mu- cells) as well as cytoplasmic mu-bearing pre-B cells and surface mu-bearing B lymphocytes. An accompanying increase in proliferative activity was indicated by the numbers of 14.8+ mu- cells and pre-B cells which accumulated in metaphase after inducing mitotic arrest with vincristine. These effects were all abrogated either by treating mice with silica to depress macrophage function or by splenectomy. In mice given multiple injections of SRBC for 4 weeks the elevated levels of early precursor B cell production and B cell genesis were sustained. The work demonstrates that the in vivo production of early precursor B cells, putatively including those at the stage of Ig heavy chain gene rearrangement, can be stimulated by exposure to external agents acting indirectly by a silica-sensitive, spleen-dependent mechanism. The findings suggest that the level of pro-B cell proliferation and primary B cell genesis normally taking place in mouse bone marrow may reflect the level of exposure to potential stimulants in the external environment mediated by activation of splenic macrophages. The possibility that abnormally high levels of macrophage activation could predispose to dysregulations of the B cell lineage is raised.     

Temporal relationship of translation and glycosylation of immunoglobulin heavy and light chains.

The initial glycosylation of MPC 11 gamma 2b heavy chains occurs quantitatively in vivo when the nascent heavy chains reach a size of approximately 38 000 daltons. Nonglycosylated, completed MPC 11 heavy chains cannot be glycosylated in these cells. Other classes of mouse heavy chains (i.e., mu, alpha, and gamma 1) also appear to be glycosylated as nascent chains; nonglycosylated, completed heavy chains cannot be glycosylated by the cell in any of these cases. In contrast, variant MPC 11 cells synthesizing a heavy chain with a carboxy-terminal deletion appear to glycosylate some heavy chains prior to chain completion and some heavy chains after chain completion and release from the polysomes. Similar to the variant MPC 11 cells, MOPC 46B cells (which synthesize a kappa light chain containing an oligosaccharide attached to an asparagine located 28 residues from the amino terminus) glycosylate the majority of light chains after prior to chain completion but also some light chains after chain completion and release from the polysomes. In addition, it appears that, although completed MOPC 46B light chains can be glycosylated if they are present in a monomeric form, they cannot be glycosylated if they are present in a covalent dimeric form.     

Isolation of scid pre-B cells that rearrange kappa light chain genes: formation of normal signal and abnormal coding joins.

Consistent with an ordered immunoglobulin (Ig) gene assembly process during precursor (pre-) B cell differentiation, we find that most Abelson murine leukemia virus (A-MuLV)-transformed pre-B cells derived from scid (severe combined immune deficient) mice actively form aberrant rearrangements of their Ig heavy chain locus but do not rearrange endogenous kappa light chain variable region gene segments. However, we have identified several scid A-MuLV transformants that transcribe the germline Ig kappa light chain constant region and actively rearrange the kappa variable region gene locus. In one case progression to the stage of kappa light chain gene rearrangement did not require expression of Ig mu heavy chains; furthermore, this progression could not be efficiently induced following expression of mu heavy chains from an introduced vector. As observed in pre-B cell lines from normal mice, attempted V kappa-to-J kappa rearrangements in scid transformants occur by inversion at least as frequently as by deletion. The inverted rearrangements result in retention of both products of the recombination event in the chromosome, thus allowing their examination. scid kappa coding sequence joins are aberrant and analogous in structure to previously described scid heavy chain coding joins. In contrast, the recognition signals that flank involved coding segments frequently are joined precisely back-to-back in normal fashion. The scid VDJ recombinase defect therefore does not significantly impair recognition of, site-specific cutting at, or juxtaposition and appropriate ligation of signal sequences. Our finding that the scid defect prevents formation of correct coding but not signal joins distinguishes these events mechanistically.     

Control of IgM synthesis in the murine pre-B cell line, 70Z/3'

The murine 70Z/3 tumor resembles a pre-B cell in synthesizing only intracellular mu-chains and no detectable light chain. However, one kappa gene is already rearranged, and after overnight incubation with lipopolysaccharide (LPS), most of the cells are induced to synthesize light chain. The induced cells display IgM on their surface, but do not secrete IgM. Thus, 70Z/3 cells resemble cells poised at the pre-B cell/B lymphocyte border. We have examined synthesis and post-translational modification of mu-chains in uninduced and induced 70Z/3 cells. Isolation of mu-chains and peptide maps demonstrated that both populations synthesize intracellular forms that correspond to membrane-specific mum and secretion-specific mus chains. These intracellular forms have completed only the first of the two glycosylation steps characteristic of eukaryotic cells. After induction by LPS, L chain synthesis commences, mum and mus synthesis are both increased twofold to threefold (due to an increased rate of synthesis rather than decreased degradation), and both complex with L chain to form mu2L2 tetramers. Furthermore, the glycosylation of a subset of the mum chains is completed, and these are placed on the membrane. However, unglycosylated mu2L2 tetramers can be placed on the membrane, so glycosylation is not a requirement. These data suggest that L chain may not be sufficient for externalization of mum and mus chains. These data support the idea that the controls of membrane placement and secretion of mu chains are post-translational and that different mechanisms operate for mum and mus chains.