B cell maturation factor (BMF): a lymphokine or family of lymphokines promoting the maturation of B lymphocytes

Two in vitro B cell tumor lines have been used to characterize and partially purify a lymphokine, or family of lymphokines, from monoclonal helper T cell immune response supernatants. These lymphokines induce the pre-B-like 70Z/3 tumor cell to synthesize Ig L chains and express complete Ig molecules on its cell surface, and cause the mature B cell-like WEHI-279 tumor cell to increase its ratio of secretory to membrane mu production, begin high rate Ig secretion, and then die. Most of the activity responsible for these changes co-purifies during five different separation procedures, implying the existence of a discrete molecule or closely related class of molecules able to mediate all of these effects. The molecules active in these systems appear distinct from the other lymphokines IL 1, IL 2, G/M-CSA, TRF, IFN, BCGF, and the activity variously termed IL 3/BPA/PSF/HCGF/MCGF, etc. We call these B cell-differentiating molecules BMF, or B cell maturation factor(s). The BMF molecules are mildly acidic (pI 5 to 6 in various conditions), extremely hydrophobic, probably heterogeneously glycosylated glycoproteins, with an apparent m.w. of 50,000 to 55,000 by gel permeation chromatography and 16,000 by SDS-PAGE. BMF has been purified approximately 3000-fold by three sequential chromatographic steps, with the use of the B tumor line assay systems. BMF molecules thus purified also cause normal resting splenic B cells to mature to the state of active Ig secretion.     

Physicochemical and functional properties of murine B cell-derived B cell growth factor II (WEHI-231-BCGF-II)

A murine B lymphoma cell line, WEHI-231, constitutively secreted a kind of B cell stimulatory factor (BSF) that induced proliferation and IgM secretion in splenic B cells as well as BCL1 cells. Growth- and differentiation-promoting activities were not separated by various kinds of chromatographies on the basis of the m.w., isoelectric point, or hydrophobicity, and the degree of both activities in crude supernatants, DEAE-Toyopearl, TSK-3,000SWG, Mono P, and Phenyl-5PW fractions increased in a dose-dependent manner with complete correlation. The partially purified factor (WEHI-231-BGDF) did not show any other activities, such as IL 1, IL 2, interferon, or colony stimulating factor. WEHI-231-BGDF induced proliferation and IgM secretion in activated B cells with low density, but not in small resting B cells. WEHI-231-BGDF showed synergistic effect with dextran sulfate but not with anti-Ig in the induction of proliferation or IgM secretion in small resting B cells. WEHI-231-BGDF did not show any effect on Xid B cells. The relationship with several T cell-derived BSF and the significance of B cell-derived BSF in the B cell responses are discussed.     

Distinction of B cell maturation factors from lymphokines affecting B cell growth and viability

Supernatants from S26.5 helper T cells, autoimmune viable motheaten (mev/mev) mouse spleen cells, EL4 lymphoma cells, and recombinant DNA-derived interferon gamma (IFN-gamma), all of which display B cell maturation factor (BMF) activity, were assayed for effects on B cell proliferation alone and with Dextran Sulfate (DxS) and anti-immunoglobulin antibodies (alpha-Ig). Both EL4 and S26.5 supernatants showed BCGF-II (DxS co-stimulator) activity, whereas only EL4 supernatant had BCGF-I (alpha-Ig co-stimulator or BSF-I) activity. Supernatants from mev/mev spleen cells and recombinant DNA-derived IFN-gamma showed no activity in either assay. Fractionation of S26.5 supernatant by chromatofocusing showed a divergence of BMF activity (BMF-T, pIa of 6.0) from BCGF-II activity (pIa of 5.4), providing evidence for their physical nonidentity. IFN-gamma, which decreases B cell viability in culture, was separable from BMF-T by phenyl-Sepharose chromatography. BMF-T from S26.5 supernatant was separated from IFN-gamma and BCGF-II and was shown to induce B cell maturation without affecting B cell proliferation. The molecular characteristics of the purified BMF-T were pIa 6.0, Mr 55,000 by G-75 gel filtration, and Mr 16,000 by SDS-PAGE. These data demonstrate that several lymphokines (BMF) exist that mediate the maturation of B cells to active Ig secretion without stimulating B cell proliferation.     

The requirement for surface Ig signaling as a prerequisite for T cell:B cell interactions. A possible role for desialylation

Models for T cell:B cell collaboration suggest that activated B cells process and present Ag to Th cells which subsequently induce B cell proliferation and differentiation. In contrast to activated B cells, resting B cells have generally been shown to be less efficient APC. If this model of T:B collaboration is physiologically correct, then resting B cells must undergo a phenotypic change that permits effective interaction with T cells. In this report, the requirement for rapid signaling through surface Ig on resting B cells for the induction of T:B interaction was investigated with an in vitro clustering assay. Resting splenic B cells were unable to form specific conjugates with T cell clones, unless the B cells were first treated with neuraminidase to remove sialic acid. In contrast, LPS-activated B cells were able to form conjugates without prior treatment. The ability of antibody against LFA-1 or L3T4 to inhibit cluster formation depended on the state of B cell activation in that anti-LFA-1 and anti-L3T4 mAb inhibited cluster formation by neuraminidase-treated resting B cells, but not by LPS-activated B cells. In addition, Ag-specific B cells which were isolated by their capacity to bind specific Ag were able to form clusters without any additional treatment. Moreover, treatment of resting splenic B cells with anti-mu-antibody induced clustering potential in B cells in as little as 10 min, suggesting that signaling through surface Ig was sufficient to induce this phenotypic change in B cells. Furthermore, activation of protein kinase C and Ca2+ mobilization were shown to be involved in that PMA and ionomycin treatment were also able to induce clustering potential in resting B cells. The rapid induction of clustering potential in resting B cells after signaling through surface Ig may represent a fundamental change in B cell physiology which occurs after recognition of specific Ag and may be required for effective cognate recognition between resting hapten-specific B cells and carrier-specific T cells. The potential role of desialylation for the induction of T:B interaction is discussed.     

A "lymphokine-like" soluble product that induces proliferation and maturation of B cells appears in the serum-free supernatant of a T cell hybridoma as a consequence of mycoplasmal contamination

The serum-free supernatant of a cloned murine T cell hybridoma supports the proliferation and maturation to Ig secretion of purified B cells (mu+ cells) from BALB/c nu/nu mice, but has no effect on the proliferation of nylon wool-selected BALB/c nu/+ splenic T cells. Although the supernatant activates B cells without co-stimulation, it synergizes with anti-mu for the proliferative response. The induction of B cell proliferation and maturation to Ig secretion is directly related to contamination of the hybridoma by Mycoplasma hyorhinis. Hybridoma cells freed of mycoplasma by detergent treatment fail to produce active supernatant, and reinfection of the treated cells reconstitutes the activity. Furthermore, deliberate infection of a mycoplasma-free unrelated T cell hybridoma, as well as the monocytic cell line P388D1, results in the production of supernatants with B cell proliferating activity. Mycoplasma organisms isolated from the supernatant induce B cell proliferation without subsequent maturation to Ig secretion. Gel filtration chromatography of the supernatant from mycoplasma-contaminated hybridoma cells yields two peaks of activity. The first peak, found at the exclusion limit of the gel, results in B cell proliferation without maturation and may be attributed to mycoplasma organisms. The second peak (average m.w. 90,000) results in B cell proliferation as well as differentiation to Ig secretion. A "lymphokine-like" soluble product released by Mycoplasma hyorhinis is most likely responsible for this B cell activation, because fractionation of the supernatant from deliberately contaminated P388D1 cells gives essentially the same results, and gel filtration of mycoplasma-free supernatants does not generate any active fractions. The possibility should be considered that mycoplasma-derived soluble products may be among the many factors controlling in vitro B cell growth and maturation.     

Bursectomy of chicken embryos at 60 hours of incubation leads to an oligoclonal B cell compartment and restricted Ig diversity

Chickens that have been surgically bursectomized at 60 h of embryonic development usually generate Ig producing B cells; however, the bursectomized chickens are incapable of specific antibody responses, even after repeated immunization. In the present work, we analyzed the molecular basis of this immunodeficiency. In the bursectomized chickens, DNA sequencing revealed a repertoire of Ig L and H chains with a low number of different V-J and V-D-J joints, indicating an oligoclonal B cell compartment. In addition, the L and H chains belonging to each B cell clone had similar gene conversion events in the V region. In situ hybridization to Harderian gland tissue sections showed, that B cells of the bursectomized chickens were, however, capable of terminal plasma cell maturation. Thus, in chickens that were lacking the bursal microenvironment, 1) only a few B cell precursors differentiated into mature Ig-producing B cells, 2) low rate of gene conversion resulted in restricted Ig diversity. Regarding the chicken B cell differentiation, the present data support a model that the induction of B cell differentiation is a bursa-independent event, whereas the bursa of Fabricius has a crucial role in the amplification and diversification of the embryonic B cell repertoire.     

Gamma-interferon induces apoptosis of the B lymphoma WEHI-279 cell line through a CD95/CD95L-independent mechanism.

Gamma-interferon (IFN-gamma) a cytokine produced by CD4+ T helper type 1 cells, CD8+ T cells and natural killer (NK) cells, plays a central role in the development of humoral and cell-mediated immunity. IFN-gamma participates in the maturation and differentiation of B cells, but it has been previously reported that IFN-gamma may inhibit the early stages of B cell activation. We report that the inhibition of the B lymphoma cell WEHI-279-proliferation induced by IFN-gamma, involves the induction of typical features of apoptosis (nuclear chromatin condensation and fragmentation, cell shrinkage, phosphatidyl-serine (PS) exposure and mitochondrial membrane potential (delta psim) loss). IFN-gamma-mediated B cell apoptosis was decreased by the addition of the T helper type 2 cytokine, IL-4. WEHI-279 cells express CD95 and undergo apoptosis after treatment with either an agonistic anti-CD95 Ab or with a soluble recombinant CD95L. However, incubation with CD95-Fc or TRAIL-R1-Fc fusion proteins, did not prevent IFN-gamma-mediated apoptosis, suggesting that IFN-gamma-mediated apoptosis occurs independently of CD95/CD95L and TRAIL-R/TRAIL interactions. IFN-gamma-mediated apoptosis is associated with caspase-3 activation that can be prevented by the addition of the broad caspase inhibitor zVAD-fmk. These data indicate that IFN-gamma may play a major role in the regulation of B cell apoptosis, and suggest the involvement of an alternative pathway which is independent of the death receptors.     

Building and operating an antibody factory: redox control during B to plasma cell terminal differentiation

When small B lymphocytes bind their cognate antigens in the context of suitable signals, a dramatic differentiation program is activated that leads to the formation of plasma cells. These are short-lived specialized elements, each capable of secreting several thousands antibodies per second. The massive increase in Ig synthesis and transport entails a dramatic architectural and functional metamorphosis that involves the development of the endoplasmic reticulum (ER) and secretory organelles. Massive Ig secretion poses novel metabolic requirements, particularly for what concerns aminoacid import, ATP synthesis and redox homeostasis. Ig H and L chains enter the ER in the reduced state, to be rapidly oxidised mainly via protein driven relays based on the resident enzymes PDI and Ero1. How do plasma cells cope with the ensuing metabolic and redox stresses? In this essay, we discuss the physiological implications that increased Ig production could have in the control of plasma cell generation, function and lifespan, with emphasis on the potential role of ROS generation in mitochondria and ER.     

Effect of B cell differentiation factor (BCDF) on biosynthesis and secretion of immunoglobulin molecules in human B cell lines

Stimulation of human B lymphoblastoid cell lines, CESS and SKW6-CL4 with BCDF induced an increase in IgG- and IgM-secreting cells, as well as a slight increase in IgG and IgM expression on their respective surfaces. Biosynthetic labeling demonstrated that BCDF stimulation induced an increase in synthesis of secretory gamma- and mu-chains, as well as their precursors. A slight but significant increase in synthesis of membrane-bound gamma- and mu-chains and their precursors was also observed by BCDF stimulation. However, an increase in synthesis of secretory heavy chains was much higher than that in membrane-bound heavy chains in both cell lines. Pulse-chase experiments showed that increased synthesis of secretory heavy chains was not due to a decrease in degradation. BCDF stimulation induced a preferential increase in mRNA specific for secretory gamma- and mu-chains in CESS and SKW6-CL4 cells, respectively. These results suggest that BCDF induces an increase in the level of mRNA specific for secretory heavy chains, and then induces final maturation of B cells into immunoglobulin-secreting cells.     

Bone marrow stromal cell regulation of B-lymphopoiesis. I. The role of macrophages, IL-1, and IL-4 in pre-B cell maturation

Our experiments have addressed regulation of B lymphocyte formation by bone marrow stromal cells. Stromal cells appear to produce a regulatory factor that acts at the pre-B cell stage to induce the expression of Ig L chains and surface Ig. Bone marrow stromal cell conditioned medium was found to contain this factor and the active component was partially purified by HPLC. This stromal cell-derived factor had a m.w. between 16,000 and 20,000, was specifically neutralized by anti-IL-4 mAb, 11B11, and enhanced the proliferation of anti-mu-stimulated B cells. We also found that rIL-4 induced B cell formation in culture. In our studies, IL-1 had no direct effect on pre-B cell maturation, however, IL-1 was found to stimulate the production of IL-4 by both heterogeneous bone marrow stromal cells and a cloned stromal cell line, SCL-160. These effects of IL-1 on factor production by stromal cells were duplicated by the addition of bone marrow-derived macrophages to SCL-160 cells. We conclude that stromal cell-derived IL-4 is a physiologic stimulator for B cell generation. In addition, macrophages appear to play a role in B cell formation by regulating the production of IL-4 by stromal cells via the secretion of IL-1.     

Somatic cell hybrids of mouse myeloma cells and B lymphocytes from a patient with agammaglobulinemia: failure to secrete human immunoglobulin.

Somatic cell hybrid clones were isolated from the fusion of RPC5.4 mouse myeloma cells and B lymphocytes from a patient with common varied agammaglobuinemia. The patient has B lymphocytes that synthesize immunoglobulin but fail to secrete immunoglobulin. The hybrid character of the six clones was established by examination of metaphase chromosome spreads. Most of the hybrid clones expressed mouse and human surface immunoglobulin. All of the clones synthesized immunoglobulin of mouse and human parental origin. Mouse parental immunoglobulin was secreted, whereas the human parental immunoglobulin was not secreted. Human light chain molecules were secreted as part of hybrid H2L2 molecules formed with mouse heavy chains. Human heavy chains had a reduced m.w. in comparison to the mouse heavy chains. Kinetic experiments indicated that human Ig was synthesized in amounts that were comparable to the mouse Ig. Pulse-chase experiments showed that that the intracellular human Ig was removed from the cytoplasm, probably by degradation. These experiments demonstrate that the hybrid cells are an in vitro model of naturally occurring failure of immunoglobulin secretion from agammaglobulinemia. The failure of fusion with mouse myeloma cells to complement the secretion defect suggests that these B cells produce an altered immunoglobulin molecule that is not programmed for secretion.     

Role of DNA synthesis in secretion of immunoglobulin from murine B cells stimulated by T cell derived lymphokines

We have investigated whether cell division is required for induction of Ig secretion from three types of B cells, which represent distinct activation states: normal splenic B cells, anti-Ig-treated B cells, and a monoclonal murine B cell tumor, BCL1. Polyclonal Ig secretion was stimulated in vitro by LPS or by lymphokines produced by EL-4 cells (EL-4 SN), which includes B cell growth factor II (BCGF II). LPS and EL-4 SN were mitogenic for all three cell populations and stimulated substantial IgM secretion from both B cells and anti-Ig blasts. Aphidicolin, a reversible inhibitor of DNA synthesis, abolished IgM secretion from B cells and anti-Ig blasts induced by either mitogen, indicating that Ig-secreting cells in these cultures are part of a cycling population. BCL1 tumor cells respond to BCGF II (but not to interleukin 2 or B cell stimulatory factor 1) with IgM secretion and cell division, allowing a direct assessment of the influence of BCGF II-stimulated cell division on secretion of IgM. Secretion by these cells during the first 24 hr of culture was not substantially affected by aphidicolin, but secretion at 48 or 72 hr was markedly inhibited. Culture of BCL1 cells for 48 hr with aphidicolin alone had no effect on cell viability or on subsequent responsiveness if the drug was removed, eliminating non-specific toxicity as an explanation of the drug's effect. Addition of aphidicolin during the last 24 hr of culture to either normal B cells or BCL1 cells was much less effective at inhibiting IgM secretion. These results indicate that the cells that secrete IgM in response to BCGF II also synthesize DNA when exposed to this factor. Thus, induction of high-rate Ig secretion from murine B cells by some stimuli, including BCGF II, may require at least one round of cell division.     

CD5+ peritoneal B cells express high levels of membrane, but not secretory, C mu mRNA.

We used in situ hybridization to study Ig mRNA levels in murine peritoneal and splenic B cells. Ig mRNA production fell into three distinct groups: low, intermediate, and high. Splenic B cells primarily exhibited low levels characteristic of resting B cells or high Ig mRNA levels characteristic of plasma cells. In contrast, a significant fraction of peritoneal B cells exhibited intermediate Ig mRNA levels. Intermediate Ig mRNA was T cell dependent in that congenic nu/nu mice had far fewer peritoneal cells expressing the intermediate Ig message than their wild type counterparts. CD5+ CD11b+ IgMbright+ peritoneal B cells were found to be mainly responsible for the production of intermediate Ig mRNA levels. The peritoneal CD5- CD11b+ IgMbright+ "sister" B cell subpopulation contained a lower percentage of intermediate Ig mRNA-producing B cells. CD5-CD11b-IgMdull+ "conventional" B cells produced negligible levels of Ig mRNA, comparable to those of unfractionated splenic B cells. Northern analysis showed that the majority of Ig mRNA expressed in the peritoneum is of the membrane rather than the secreted form. Consistent with that result, in short-term culture, peritoneal cells showed markedly less Ig secretion than did spleen cells. These studies describe novel Ig mRNA expression by peritoneal B cells and emphasize that within the peritoneal cavity, B cells do not tend to become antibody-secreting cells.     

Pre-exposure of human B cells to recombinant IL-1 enhances subsequent proliferation

The reported effects of the monocyte-derived cytokine IL-1 on human B lymphocytes are both varied and controversial. IL-1 has been reported to augment both proliferation and Ig secretion of previously activated human B cells. In the present study highly purified splenic B cells were cultured with rIL-1 before, simultaneously with, and after the addition of the polyclonal B cell mitogen, anti-Ig. rIL-1 had no significant effect on B cell proliferation when added simultaneously with or after B cell activation with anti-Ig. However, incubation of splenic B cells with rIL-1 for 24 h before stimulation with anti-Ig appeared to enhance mitogenesis. With the observation that rIL-1 exerted effects on resting B cells, the effect of rIL-1 on several events which accompany B cell activation was examined. rIL-1 failed to stimulate RNA synthesis, effect increases in cell size or intracellular Ca2+ levels, or lead to the hyperexpression of MHC class II or B cell activation Ag. These studies suggest that rIL-1 does not activate B cells but primes them to respond to subsequent activation.     

Predominance of nonproductive rearrangements of VH81X gene segments evidences a dependence of B cell clonal maturation on the structure of nascent H chains

Ig H chain V regions using the VH81X gene segment were PCR amplified from genomic DNA obtained from either splenic B cells or surface (s)Ig- bone marrow cells of BALB/c mice. Sequence analysis demonstrated that 93% of VH81X containing H chain V region genes in splenic B cells were rearranged nonproductively. Furthermore, 74% of rearrangements of VH81X among sIg- bone marrow cells were nonproductive. This contrasts with previous results obtained for rearrangements of members of the VH36-60 gene segment family among sIg- cells wherein, as a consequence of extensive clonal expansion after productive H chain V gene rearrangement, 80% of rearrangements were productive. The low proportion of productive rearrangements of VH81X is interpreted as indicating that most productive rearrangements of VH81X cannot facilitate clonal expansion, which would support the hypothesis that selection for clonal expansion and maturation is dependent on the amino acid sequence of nascent H chains. Additionally, because most productive rearrangements of VH81X cannot facilitate clonal maturation but do appear to mediate allelic exclusion, these processes are likely to be regulated independently.