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.     

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.     

Differential expression of the c-myb proto-oncogene marks the pre-B cell/B cell junction in murine B lymphoid tumors

A series of murine B lymphoid tumor cell lines which are representative of the pre-B cell, immature and mature B cell, and plasma cell stages of B cell development have been examined for expression of c-myb proto-oncogene mRNA. The pre-B cell lymphoma cell lines express equivalent high steady state levels of c-myb mRNA. In contrast, the B cell lymphoma and plasmacytoma cell lines express steady state c-myb mRNA at levels which are 0.005 to 0.1 times that of the pre-B cell lymphoma lines. These results correlate high levels of c-myb mRNA expression with the pre-B cell stage of development. Subclones of the 1881 pre-B cell lymphoma which express K light chain and are surface IgM-positive as well as two types of hybrid B lymphoid cell lines have been used to demonstrate that surface immunoglobulin expression is not sufficient to result in the down-regulation of c-myb mRNA levels or changes in the expression N-myc mRNA, lambda 5 mRNA, or the BP-1 surface antigen which are markers of the pre-B cell stage of development. Thus, changes in the expression of genes which are independent of immunoglobulin expression are associated with transition from the pre-B cell to the immature B cell stage of development.     

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.     

Role of T cell-replacing factor (TRF) in the murine B cell differentiation: induction of increased levels of expression of secreted type IgM mRNA

T cell-replacing factor (TRF) is known to play a critical role in the regulation of B cell growth and differentiation. In this study, the role of TRF in the expression of mRNA for both IgM and IgG1 class was investigated. The TRF was purified from cellfree supernatants from a T cell hybridoma, B151K12. RNA was isolated from chronic B cell leukemia (BCL1) cells, DNP-KLH-primed B cells, or normal B cells cultured with or without LPS, and LPS plus TRF or LPS plus BSF-1. The steady state level of isotype-specific mRNA was assessed by Northern blot analysis with a mu-specific or a gamma 1-specific probe. It was demonstrated that BCL1 and purified B cells cocultured with TRF expresses increased levels (twofold and fourfold, respectively) of secreted forms of mu mRNA. Purified B cells from DNP-KLH-primed mice also expressed increased levels (twofold to fourfold) of mu as well as gamma 1 mRNA for secreted form by stimulation with TRF. Total expression of mu mRNA, however, was approximately threefold higher than that of gamma 1 mRNA. The stimulation of normal B cells with LPS plus TRF induced an increase in the levels of mu mRNA and gamma 1 mRNA expression, fourfold and threefold, respectively. However, the levels of gamma 1 mRNA expression was one-third of that induced in B cells stimulated with LPS plus BSF-1. These results indicate that TRF preferentially induces increased levels of secreted type of mu mRNA and induces less gamma 1 mRNA than BSF-1. The differential role of TRF from BSF-1 in the expression of Ig mRNA will be discussed.     

Polyadenylate-deficient analogues of poly(A)-containing mRNA sequences in cultured AKR mouse embryo cells

Five to six percent (by mass) of AKR-2B mouse embryo cell polysomal RNA consists of messenger RNA sequences which may exist in polyadenylated form. In the steady state, however, only 30–40% of these molecules are retained by extensive passage over oligo(dT)-cellulose, the remainder being present in the form of poly(A)-deficient analogues. Within experimental limits, these poly(A)-deficient analogues contain representatives of all poly(A)-containing mRNA sequences in these cells. An analysis of the kinetics of hybridization of cDNA probes enriched for either abundant or rare poly(A)-containing mRNA sequences suggests that the frequency distributions of poly(A)-containing and poly(A)-deficient analogues are dissimilar, and that a relationship exists between the intracellular frequency of a given mRNA sequence and the number of poly(A)-deficient analogues of that sequence. High frequency sequences appear to be enriched in the poly(A)-containing fraction, while low frequency sequences are predominately associated with the poly(A)-deficient fraction, thus, poly(A) may play a role in the regulation of mRNA frequency in the cytoplasm.     

Resonance energy transfer study using a rhenium metal-ligand lipid conjugate as the donor in a model membrane.

We measured steady state and time-resolved resonance energy transfer between donors and acceptors in model membranes. The donor was a long lifetime rhenium-lipid complex, which displayed a mean lifetime of 1 microsecond and lifetime components as long as 3 microseconds in the labeled DOPC membranes. The transfer efficiencies were found to be substantially larger than those predicted without consideration of lateral diffusion. The larger transfer efficiencies are consistent with a mutual lateral diffusion coefficient in the membrane near 2 x 10(-8) cm2/s. These results demonstrate that lateral diffusion in membranes can be detected with microsecond lipid probes.     

Processing at immunoglobulin polyadenylation sites in lymphoid cell extracts.

We have developed an in vitro system for polyadenylation of RNA substrates in cell-free nuclear extracts prepared from murine cells of lymphoid origin. RNA substrates containing the adenovirus L3, murine immunoglobulin (IgM) secreted and membrane polyadenylation sites were accurately polyadenylated in these extracts. Kinetic analysis showed that the rate of polyadenylation in vitro responds proportionally to the substrate concentration. Quantitation of the initial rate of polyadenylation at the three sites permitted comparison of the activities of extracts prepared from HeLa cells, B cells (Wehi 231) and plasmacytoma cells (P9.37.11). From this analysis, we concluded that in all three extracts the polyadenylation activity at the L3 site was higher than that of either of the IgM sites. In contrast to the preferential utilization of the secreted site in vivo in plasmacytomas, this site was not selectively processed in plasmacytoma as compared to B cell extracts. The efficiency of polyadenylation at both IgM sites in the plasmacytoma extract was significantly lower than that in the B cell extract. The common low activity at the IgM sites in the plasmacytoma cell extract suggests that the rate-limiting step for polyadenylation at these two sites differs from that at the L3 site.     

Effect of upstream RNA processing on selection of mu S versus mu M poly(A) sites.

All of the regulatory factors responsible for augmenting microseconds mRNA levels preceding the dramatic increase in secretory IgM production upon B cell activation has not been totally elucidated. Whereas previous experiments have centered on the region of the gene specifying the choice between splicing to mu M exons versus selection of the mu S poly(A) site, we have found that upstream sequences within the Cmu gene, specifically the Cmu 4 acceptor splice site together with intronic sequences between the Cmu 3++ and Cmu 4 exons, play an important role in dictating the precision or the extent of splicing to the mu M exons even under conditions in which functional polyadenylation factors should be in excess. Therefore, splicing of upstream exons can affect remotely located downstream exons. These findings suggest that regulation of differential mu S/mu M mRNA expression may involve general processing enzymes that recognize specific cis -regulatory sequences residing within the body of the mu gene and account for the unique ability of activated B cells to secrete copious amounts of IgM.