Differential effects of interleukin 2 vs B cell growth factor on human B cells

The effects of recombinant interleukin 2 (IL-2) and high m.w. (HMW) B cell growth factor (BCGF) were examined on normal human peripheral blood B cells activated with Staphylococcus aureus Cowan I (SAC). When SAC-activated B cells were separated into Tac-antigen (Tac-Ag)+ and Tac-Ag- fractions by a cell sorter, recombinant IL-2 induced only the Tac-Ag+ cells to proliferate, whereas both Tac-Ag+ and Tac-Ag- cells responded to HMW-BCGF (m.w. 60,000). Alternatively, SAC-activated B cells were separated according to density into three fractions: low density (large) cells (82 +/- 15% Tac-Ag+), intermediate density (medium) cells (45 +/- 13% Tac-Ag+), and high density (small) cells (less than 5% Tac-Ag+). Recombinant IL-2 enhanced proliferation of low density cells the most, intermediate density cells less, and high density cells not at all. HMW-BCGF induced all three fractions to proliferate to approximately the same degree. Finally, the effects of IL-2 and BCGF on the DNA and RNA content of the various fractions of B cells was examined. RNA content was greater in IL-2-stimulated B cells than BCGF-stimulated B cells, whereas DNA content was the same in both cell populations. IL-2 and BCGF may preferentially interact with different subpopulations of B cells. The interaction of IL-2 or BCGF with normal activated B cells may induce both similar and different intracellular events.     

B cell growth-promoting activity of recombinant human interleukin 4

Human interleukin 4 (IL-4), also known as B cell stimulatory factor 1, is a T cell-derived glycoprotein consisting of 129 amino acids for which a cDNA has been recently isolated. IL-4 displays little or no B cell growth factor (BCGF) activity in the standard anti-IgM costimulatory assay using suboptimal concentrations of soluble anti-IgM antibody whereas the low m.w. BCGF is very active. When insolubilized anti-IgM was used as the costimulating agent, both IL-4 and the low m.w. BCGF were found to promote B cell proliferation. Human IL-4 is able to induce the proliferation of B lymphocytes preactivated for either 1 day with insolubilized anti-IgM antibody or for 3 days with Staphylococcus aureus strain Cowan I. However, IL-4 is poorly mitogenic for B cells preactivated for 1 day with the Staphylococcus strain whereas the low m.w. BCGF strongly enhances the proliferation of these B cells. These two findings demonstrate that the preactivation signal necessary to induce human B cells to proliferate in response to IL-4 is critical. The increased tritiated thymidine ([3H]dThd) uptake in preactivated B cell cultures with IL-4 reflects cel proliferation because cell cycle analysis demonstrates that IL-4 induces activated B cells to enter the S and G2/M phases of the cell cycle and the addition of IL-4 to preactivated B cell cultures permits the recovery of three- to fourfold more B cells after 4 days of culture. IL-4 and the low m.w. BCGF act in concert to induce the proliferation of anti-IgM-preactivated B cells as demonstrated by [3H]dThd uptake and cell cycle analysis. In striking contrast to the demonstrated antagonistic effect of interferon-gamma on the IL-4-induced expression of the low affinity receptor for IgE (Fc epsilon RL/CD23), on B cells, it was found that interferon-gamma enhanced the IL-4-induced proliferation of anti-IgM-preactivated B cells. Finally, it was found that IL-4 had to be present continuously during the culture period to exert an optimal growth-promoting effect on B cell blasts. As a conclusion, IL-4 is able to induce the proliferation of an appropriately activated subpopulation of human B cells.     

Characterization of a T4+/Leu-8+ T cell clone that directly helps B cell Ig production by secreting B cell differentiation factor

A human T4+/Leu-8+ T cell clone (YA2) was established by phytohemagglutinin activation and interleukin 2 (IL 2) propagation. Functional characterization of this clone demonstrated that it provided potent help towards Ig production by pokeweed mitogen-stimulated B cells in the presence of small numbers of autologous T cells or by Staphylococcus aureus Cowan I (SAC)-activated B cells in the presence of B cell growth factor (BCGF). YA2 provided no help to resting B cells and minimal help to either unactivated B cells cultured with BCGF or SAC-activated B cells. Supernatant generated from clone YA2 by IL 2 stimulation had significant B cell differentiation activity but no BCGF or IL 2 activity. Thus, YA2 is a T4+/Leu-8+ potent direct helper only to B cells that are activated and proliferating due to its selective secretion of a differentiation factor, and not an activation and growth factor. The availability of phenotypically defined cloned populations of T cells with restricted functional helper activity related to the secretion of selected B cell tropic factors should prove useful in the dissection of the role of individual T cell subsets in the regulation of the human B cell cycle.     

Production of B cell growth factor by normal human B cells

Although it has been demonstrated that malignant human B cell lines are capable of producing B cell growth factor (BCGF), production of BCGF by normal B cells has not been shown. In this study, we demonstrate BCGF production by normal B cells, achieved by using human peripheral blood B cells prepared by a positive selection technique and stimulated with Staphylococcus aureus Cowan I (SAC) for 12 hr. SAC was removed from the supernatants by anti-SAC-coupled Sepharose. Supernatants absorbed with this antibody were functionally free of SAC, as demonstrated by their inability to activate resting B cells. B cells stimulated with SAC for 12 hr produced BCGF activity that was generally unmeasurable in supernatants by 36 hr. Characterization of BCGF produced by SAC-stimulated B cells revealed a m.w. of 32,000 by high-performance liquid chromatography sieving and sodium dodecyl sulfate-polyacrylamide gel electrophoresis; this BCGF was found to have an isoelectric point of 6.7. Furthermore, this BCGF lacked interleukin 1, interleukin 2, interferon, and B cell differentiation factor activity. This observation that BCGF can be produced by normal human B cells is significant because it demonstrates for the first time that normal B cells have the ability to provide their own growth factors or the growth factors for other B cells.     

Human recombinant IL-3 stimulates B cell differentiation

To investigate the role of human IL-3 in B cell differentiation, we examined its effect on IgG secretion from normal B cells and a B cell line, JDA. The effect of IL-3 was compared to that of IL-6. IL-3 stimulated IgG secretion from tonsil B cells or peripheral blood-derived B cells activated by Staphylococcus aureus Cowan I strain. This effect required the presence of IL-2. Neither B cell growth factor (BCGF) nor IFN-gamma replaced IL-2 in this function. IL-6 stimulated similar IgG secretion from tonsil B cells and also required the presence of IL-2. Moreover, the combination of IL-3 and IL-6 induced IgG secretion equivalent to that induced by either lymphokine. These data suggest that IL-3 and IL-6 might affect normal B cell differentiation by similar mechanism(s). The IL-3 effect on B cells appears to be caused by direct interaction with B cells because IL-3 induced a dose-dependent stimulation of IgG secretion from the JDA cells. This stimulation did not require the presence of IL-2. IL-6 displayed a similar effect on JDA cells and did not require IL-2. However, when IL-3 was combined with IL-6 a synergistic IgG secretion was observed in JDA cultures. These data suggest that IL-3 may potentiate the human immune response via stimulation of B cell differentiation and that its effect is dependent on the target B cell population, its stage of activation and/or maturation.     

Human B cell proliferation in response to IL-4 is associated with enhanced production of B cell-derived growth factors

To investigate the capacity of human IL-4 to function as a B cell growth factor (BCGF), we studied its ability to promote proliferation of a selected B cell line. We show that the cell line, designated A4, proliferated in response to IL-4 in a dose-dependent manner. The A4 cells also proliferated in response to their own B cell derived growth factor (B. BCGF), suggesting autocrine-mediated growth. The ability of IL-4 to induce proliferation of the A4 cell line was dependent on the level of autocrine growth. At low cell density, IL-4 induced marked dose-dependent proliferation. However, as A4 cell density increased, the ability of IL-4 to induce proliferation was diminished. The possibility that IL-4 may be mediating the autocrine growth of A4 cells was ruled out, because A4 cell-derived BCGF failed to induce CD23/low affinity receptors for the Fc region of IgE on activated tonsillar B cells and anti-IL-4 antibody did not block B. BCGF activity. We found that IL-4 stimulation of A4 cells and activated tonsillar B cells is associated with enhanced production of B. BCGF. These data indicate that human IL-4 has the capacity to promote proliferation of the B cell line A4, and that the ability of IL-4 to function as BCGF is associated with enhanced autocrine growth of activated B cells.     

Development and characterization of a human B cell line that responds to B cell growth factor but not interleukin 2

The human lymphoblastoid cell line we present here proliferated in response to a 14,000 m.w. B cell growth factor (BCGF), and not to interleukin 2 (IL 2). This cell line, designated B-A3, was established by Epstein Barr virus (EBV) transformation of Staphylococcus aureus Cowan I (SAC)-activated spleen B cells, and has been maintained in RPMI 1640 medium complemented with 15% fetal calf serum (FCS) without the addition of other exogenous growth factors. A proliferative response, as measured by [3H]thymidine uptake of B-A3 cells was significantly induced by either commercial IL 2-free human BCGF preparations, or phytohemagglutinin-stimulated mixed lymphocyte culture supernatant at all FCS concentrations used in the assay. The most marked proliferation due to BCGF, however, was observed in the absence of FCS. This BCGF-induced proliferation was not influenced by IL 2 or interferon-gamma (IFN-gamma), because both recombinant IL 2 and IFN-gamma failed to induce proliferation. The response of B-A3 cells to a specific BCGF was additionally indicated by the responsiveness of this cell line to BCGF purified by a series of chromatographic steps. The BCGF to which B-A3 cells responded had a m.w. of 14,000 and was similar to low m.w. BCGF reported from other laboratories. Surface characterization of B-A3 cells, analyzed by flow cytometry with a panel of monoclonal antibodies, demonstrated that the majority of B-A3 cells were stained positively with Leu-12, HLA-DR, and surface IgG markers, whereas staining with surface IgM, IgD markers, pan T cell markers (Leu-4 and Leu-9), and IL 2 receptor (Tac) were consistently negative. Taken together, the human lymphoblastoid cell line we present here responded specifically to a low m.w. BCGF. This cell line may be of value in the purification of BCGF to homogeneity, in studies of the interactions of BCGF with human B cells, and in the identification of the BCGF receptor.     

Identification of a receptor for high molecular weight human B cell growth factor

Regulation of the proliferation of human B lymphocytes is under the control of several different signals. Various B cell growth factors (BCGF) have been described including a 60-kDa BCGF called high m.w. BCGF (HMW-BCGF). In this paper we describe a mAb BA5 that blocks the proliferation of normal activated human B lymphocytes in response to HMW-BCGF and does not affect the proliferation of T cells in response to PHA or IL-2. BA5 shows minimum binding to resting B cells, significantly enhanced binding to resting B cells, significantly enhanced binding to activated B cells and essentially no binding to resting or activated T cells. BA5 recognizes a 90-kDa protein from solubilized membranes of activated B cells. 125I-HMW-BCGF cross-linked to its binding site on activated B cells produces a 150-kDa R-protein complex. Unlabeled HMW-BCGF cross-linked to its binding site on activated B cells produces a 150-kDa band recognized by both BA5 and BCGF/1/C2 (a mAb to HMW-BCGF) using Western blotting. Thus, BA5 recognizes a molecule intimately associated with the receptor for HMW-BCGF which includes a binding site for HMW-BCGF. BA5 can be used to explore the role of HMW-BCGF and B cell proliferation in various aspects of human B cell physiology.     

Sequential effect of a high molecular weight B cell growth factor and of interleukin 2 on activated human B cells

A high m.w. B cell growth factor (50,000 BCGF) prepared from lectin-activated human peripheral blood lymphocyte culture supernatants acts only on B cells preactivated by a first signal. This first signal can be delivered in vitro (with anti-mu antibody (Ab)) or in vivo. Upon costimulation with anti-mu Ab the 50,000 BCGF induces an early and transient proliferative response, whereas the response to interleukin 2 (IL-2) develops more progressively. To determine the respective targets of the 50,000 BCGF and of IL-2, B cells were activated with anti-mu Ab and separated according to the expression of the IL-2 receptor (cluster designation (CD)25 antigen). CD25+ B cells do not respond to the 50,000 BCGF and do not acquire this responsiveness after an additional culture with IL-2. CD25- B cells respond to the 50,000 BCGF and not to IL-2. However, when CD25- B cells are cultured for 3 days with the 50,000 BCGF they become responsive to IL-2. These results demonstrate a pathway of B cell activation based on the ordered and sequential action of anti-mu Ab, the 50,000 BCGF, and IL-2.     

The effects of interleukin 1 on human B cell activation and proliferation

The precise role of B cell surface immunoglobulin (slg) in the activation of B cells is unclear at present. In particular, it is uncertain whether ligands interacting with the B cell slg suffice to induce proliferation, or simply induce a state of activation in which the B cell becomes responsive to growth factors made by accessory cells. We have examined the effects of two ligands, Staphylococcus aureus Cowan strain I (SAC) and antihuman mu chain (anti-mu), which interact with B cell slg on highly purified human peripheral blood and tonsillar B cells cultured at low cell concentrations. The effects on B cell proliferation of these ligands alone or in combination with highly purified interleukin 1 (IL 1) or a supernatant of a human T-T hybridoma containing a B cell growth factor (BCGF) were studied. SAC with its high cell wall content of protein A triggered maximal B cell proliferation which was not increased further by IL 1 or BCGF. High concentrations of soluble F(ab')2 fragments of goat anti-mu chain also induced significant B cell proliferation. Lower concentrations of anti-mu resulted in little or no B cell proliferation but activated the B cell to a state of responsiveness to both IL 1 and BCGF. IL 1 by itself had no effect on the proliferation of unstimulated B cells or on the proliferation of in vivo-activated B cells which responded to BCGF in vitro, but demonstrated clear synergy with low concentrations of anti-mu antibody. BCGF alone augmented the proliferation of unstimulated B cells, presumably by acting on B cells which had undergone some degree of activation in vivo. In addition, it showed marked synergy with anti-mu antibody, which resulted in proliferation similar in magnitude to that induced by SAC. This synergy was far greater than that seen between anti-mu antibody and IL 1, and the resulting proliferative response was only slightly increased by the presence of IL 1. We conclude that the importance of accessory cell factors for the initial rounds of B cell proliferation depends on the strength of the initial slg-mediated activation signal. When this is strong, the response is maximal and independent of accessory cells or accessory cell factors. When it is suboptimal, a moderate synergy is seen with IL 1 and a dramatic synergy with BCGF.     

Production of B cell-stimulating factors by B cells in patients with systemic lupus erythematosus

The production of B cell-stimulating factors (BSF) by B cells in patients with systemic lupus erythematosus (SLE) was studied in vitro. B cells from SLE patients markedly proliferated and differentiated into Ig-producing cells by in vitro culture without any stimulation. The culture supernatant of these B cells contained BSF activity that stimulated Staphylococcus aureus Cowan I-treated normal B cells to proliferate and differentiate into Ig-producing cells. By a Percoll gradient density centrifugation, BSF-producing cells were enriched in the higher density fraction, but were reduced in the lower density fraction. The BSF also stimulated the proliferation and the differentiation of SLE B cells. By a Percoll gradient density centrifugation, SLE B cells responsive to the BSF were enriched in the higher density fraction, but were reduced in the lower density fraction. The Mr of the BSF was estimated as about 18,000 Da by Sephacryl S-200 column chromatography. The BSF fraction did not possess IL-2 and IFN activity, but possessed IL-1 activity, which stimulated murine thymocyte proliferative responses. The BSF activity was partially, but not completely, absorbed by an anti-IL-1 alpha antibody. Furthermore, the BSF possessed IL-4 activity, which induced not only the proliferative responses of normal B cells stimulated with B cell mitogens, but also the expression of low affinity Fc epsilon R/CD23 on normal B cells. The BSF also possessed IL-6 activity, which induced the proliferative responses of IL-6-dependent hybridoma cells, MH-60 BSF2. Moreover, human rIL-1, rIL-4, and rIL-6 stimulated SLE B cells. These results suggest that SLE B cells spontaneously produce the BSF such as IL-1 alpha, IL-4, and IL-6 and express their receptors on their surface, and the interaction between the BSF and their receptors stimulates SLE B cells to spontaneously proliferate and differentiate into Ig-producing cells as an autocrine mechanism.     

Identification of a novel human B cell activation antigen involved in B cell growth factor-dependent proliferation

The B6.4 mAb we present here identifies a novel activation Ag of B cell lineage. The B6.4 mAb was generated by immunizing mice with B cell growth factor (BCGF)-responding BA3 cells, and selected by its capability to block BCGF-induced proliferation of BA3 cells. The inhibitory effect of this antibody on BCGF-dependent proliferation was further confirmed by using normal activated B cells in the presence of exogenous BCGF derived from T cells or B cells. Furthermore, it did not affect IL-2-dependent proliferation of B cells. The expression of the B6.4 Ag, as analyzed by FACS, is restricted to in vitro activated B cells, and remains undetectable on resting B or T cells, PHA-activated T cells, and monocytes. The B6.4 Ag is also expressed on some lymphoblastoid B cell lines and most of the lymphomas and CLL of B cell origin; however, it did not express on pre-B cell ALL and plasma cell leukemias. The B6.4 Ag has a Mr of 35,000 Da, as determined by SDS-PAGE of radiolabeled immunoprecipitates from activated B cells. The B6.4 Ag is detectable on B cells as early as 8 h after activation, and precedes that of the IL-2R or transferrin R. All of these results suggest that the B6.4 Ag is an early activation Ag of B cells and is functionally related to a receptor of BCGF, but not IL-2.     

Human recombinant IL-3 is a growth factor for normal B cells.

IL-3 is a well known hemopoietic cell growth and differentiation factor. However, its functional role in normal B cell differentiation has not been established. We have investigated the effect of IL-3 on the growth and differentiation of human B cells. IL-3 enhanced the proliferation of Staphylococcus aureus Cowan 1 strain-stimulated B cells. The optimal time of IL-3 to stimulate B cell growth was on day 2 to day 3, suggesting that IL-3 was a B cell growth factor acting in the late stage. IL-3 synergized with IL-2 to enhance B cell proliferation and differentiation. Pretreatment of B cells with IL-3 for more than 3 days increased the expression of IL-2R on B cells. However, pretreatment of B cells with IL-2 did not alter the subsequent response to IL-3, suggesting that the synergy between IL-2 and IL-3 may be attributed to the up-regulation of IL-2 response by IL-3. In addition, pretreatment of B cells with IL-4 decreased subsequent response of B cells to IL-3 as well as IL-2, suggesting that IL-3- and IL-2-responding cells passed a similar way during the early stage of B cell activation. It appears that IL-3 and IL-6 mediate normal B cell differentiation via separate mechanisms. IL-3-induced B cell differentiation was mainly mediated by increasing cell growth, whereas IL-6 induced B cell differentiation without affecting proliferation.     

Functional properties of human germinal center B cells.

Germinal centers (GCs) are histologically defined areas where B cells undergo extensive proliferation and maturation, or die of apoptosis. GC B cells isolated from human tonsils can be phenotypically identified by expression of peanut agglutinin (PNA)-binding sites and can be further divided into subpopulations based on their expression of CD77. To assess the functional potential of GC B cells, we studied CD77+ PNA+ B cells isolated from tonsils by examining their differentiation status and their ability to proliferate in vitro to various cytokines and costimulants. We found that CD77+ GC B cells are less differentiated than CD77- GC B cells; GC B cells less frequently express cytoplasmic IgG and IgM, and spontaneously secrete less Ig compared to CD77- GC B cells. To identify conditions capable of inducing GC B cell proliferation, we examined IL-4, IL-2, IFN-gamma, low molecular weight BCGF (LMW-BCGF), and an MLR supernatant along with costimulants such as anti-IgM antibody, Staphylococcus aureus Cowan I (SAC), PMA, and pokeweed mitogen (PWM). While non-GC B cells proliferate strongly in response to these stimuli, GC B cells did not proliferate. However, CD77+ as well as CD77- GC B cells mounted a rapid and strong proliferative response upon stimulation with IL-4, but only in the presence of anti-CD40 antibody. Moreover, although nine additional cytokines were examined, only IL-4 was capable of supporting CD77+ GC B cell proliferation in the presence of anti-CD40 antibody. When cells were stimulated with IL-4 and anti-CD40 antibody, we also found that IFN-gamma consistently decreased the proliferative response of CD77+ GC B cells without affecting the response of non-GC B cells. Taken together, these data indicate that GC B cells have characteristic growth requirements and that IL-4 may be important for GC B cell growth in vivo.     

The direct effects of interleukin 1, interleukin 2, interferon-alpha, interferon-gamma, B-cell growth factor, and a B-cell differentiation factor on resting and activated human B cells

A wide variety of cytokines have been demonstrated to affect B-cell function. However, it is unclear which of these mediators actually exert direct effects on the B cells themselves. In the present study, the direct role of interleukin (IL) 1, IL-2, Interferon-gamma, or Interferon-alpha in human B-cell activation, proliferation, or differentiation was examined and compared with the effects of a B-cell growth factor (BCGF) or a B-cell differentiation factor (BCDF). Highly purified human B lymphocytes were separated according to size into two nonoverlapping populations. The fraction of small B cells was incubated with IL-1, IL-2, Interferon-gamma, Interferon-alpha, BCGF, or BCDF, and cell size changes, RNA synthesis, DNA synthesis, or supernatant immunoglobulin (Ig) production were measured. Neither IL-1, IL-2, Interferon-alpha, Interferon-gamma, nor the BCGF induced substantial cell size changes, RNA synthesis, DNA synthesis, or Ig production by the small fraction of B lymphocytes; however, the BCDF could directly activate a proportion of resting B lymphocytes to secrete Ig. The fraction of large B cells was also incubated with these cytokines. While neither IL-1, Interferon-alpha, nor Interferon-gamma enhanced DNA synthesis or Ig production by the fraction of large B lymphocytes, DNA synthesis was augmented 23-fold by BCGF and IgG production was increased 7-fold by BCDF. Additionally, IL-2 slightly enhanced both proliferation and differentiation of large B cells but substantially less so than BCGF and BCDF; DNA synthesis was increased 4-fold, while Ig production in the presence of IL-2 was increased by approximately 50%. Thus, the most important lymphokines modulating the function of these two fractions of tonsillar lymphocytes were a BCGF and a BCDF.     

Reactivity of Leu-1+ tonsillar B cells to a high molecular weight B cell growth factor

This work was focused on the responsiveness of B cells from blood and tonsils to typical B cell growth factors (BCGF) in the absence of anti-mu antibody. This direct responsiveness was not observed with small dense B cells from either organ. Large tonsillar B cells but not large blood B cells did respond directly to a high m.w. BCGF (m.w. 50,000 BCGF), whereas large B cells from both organs responded directly to the low m.w. BCGF (m.w. 20,000 BCGF). The tonsillar B cells directly responsive to the m.w. 50,000 BCGF express the 4F2 marker and thus belong to a population of in vivo-preactivated B cells. Moreover, this direct responsiveness to the m.w. 50,000 BCGF is localized in Leu-1+ tonsillar B cells. The Leu-1+ and Leu-1- tonsillar B cell subsets do not differ in their responsiveness to the m.w. 50,000 BCGF upon costimulation with anti-mu antibody and to the m.w. 20,000 BCGF regardless of the presence of anti-mu antibody. Thus, the Leu-1+ subset present in tonsils shows a particular reactivity to a high m.w. BCGF.     

IgE-enhancing activity directly and selectively affects activated B cells: evidence for a human IgE differentiation factor

T cells from highly atopic individuals spontaneously secrete in vitro a factor that specifically induces IgE synthesis from normal human B cells. We investigated the effects of such T cell supernatants derived from atopic individuals (TCSN-A) on functionally distinct B cell subsets to determine at what developmental stage B cells become responsive to this IgE-enhancing activity. B cells from normal and allergic donors were separated into subsets of small resting and large activated cells by density centrifugation or unit gravity sedimentation. When stimulated by TCSN-A, large activated B cells made more IgE than small resting B cells. The difference was as much as 3300% in comparing these subsets from allergic donors. Similarly, resting B cells stimulated by Staphylococcus aureus Cowan I (SAC) made 52 to 125% more IgE in response to TCSN-A than unstimulated small resting B cells. However, IgE production from large B cells, already activated in vivo, was not enhanced by the addition of SAC. Notably, the IgE level synthesized by in vivo large activated B cells from allergic persons was markedly greater than that seen with similar cells from normal donors, whereas resting B cells purified from allergic and normal donors produced comparable levels of IgE in response to TCSN-A. These results suggest that this enhancing activity functions as an IgE differentiation factor for activated B cells. This was further confirmed by the effects of TCSN-A on the IgM- and IgE-secreting EBV-transformed human B cell line K1D5. TCSN-A specifically enhanced IgE synthesis from these cells; TCSN from normal donors, IL 2, IFN-gamma, and BCGF did not. These results confirm that this activity functions as an IgE-specific differentiation factor, directly influencing activated B cells to synthesize IgE.     

Comparative activation requirements of human peripheral blood, spleen, and lymph node B cells

Human peripheral blood, spleen, and lymph node B cells were stimulated with Cowan I Staphylococcus aureus (SA) or F(ab')2 fragments of anti-mu antibody (anti-mu) and various lymphokines and were analyzed for proliferation and generation of Ig-secreting cells (ISC). SA alone but not anti-mu stimulated minimal proliferation of each population. Recombinant IL 2 (r-IL 2) effectively promoted proliferation of SA-stimulated blood and spleen B cells, but supported less vigorous responses of lymph node B cells. By contrast, r-IL 2 enhanced DNA synthesis of all anti-mu-stimulated B cells early in culture, but did not promote sustained proliferation of anti-mu-stimulated lymph node B cells and only promoted ongoing DNA synthesis of some anti-mu-activated blood (eight out of 17) and spleen (five out of 14) B cell preparations. Recombinant interferon-gamma (r-IFN-gamma) and a commercial preparation of B cell growth factor (BCGF) also augmented DNA synthesis of all three B cell populations stimulated with SA or anti-mu early in culture, but neither alone was able to sustain maximal proliferation. Markedly enhanced sustained proliferation of all three anti-mu- and SA-stimulated B cell populations was noted when cultures were supported by the combination of r-IL 2 and BCGF, or to a lesser extent by r-IL 2 and r-IFN-gamma. The generation of ISC from SA-stimulated blood or spleen but not lymph node B cells was effectively supported by r-IL 2 alone. Differentiation of lymph node B cells required the combination of r-IL 2 and BCGF. These studies emphasize the importance of both the activation stimulus and the origin of the B cells in determining the lymphokine requirements of human B cell responsiveness.     

Demonstration that human B cells respond differently to interleukin 2 and B cell differentiation factor based on their stages of maturation

The mechanisms whereby interleukin 2 (IL 2), interferon-gamma (IFN-gamma), and B cell differentiation factor (BCDF) alone or in combination modulate human B cell differentiation are currently under intensive study. To dissect out the effects of individual lymphokines contained in mixed lymphocyte reaction-culture supernatants (MLR-CS) on B cell differentiation, we employed pure factors that possessed the same activity as factors contained in MLR-CS (IL 2: 50 U/ml, IFN-gamma: 7 U/ml, BCDF-Nal: 5 pM/ml, BCDF-YA2: 12.5% v/v) singly and in combination to human B cells. By activating purified human B cells with Staphylococcus aureus Cowan I (SAC) for 3 days, separating B blast cells by the Percoll centrifugation method, and then either using these B blast cells as B cells in the earlier stage after SAC-activation, or further culturing these B blast cells for 4 more days without any stimuli and using these B cells as B cells in the later stage after SAC-activation, we could define two different populations of cells. Disparity in the populations could be demonstrated by the observation that B cells in the earlier stage were 81.2% Tac-antigen+, 23.2% B2+, 68.9% transferrin receptor+, and 90.5% HLR-DR+, whereas B cells in the later stage were observed to be less positive for each surface antigen: 36.1% Tac-Ag+, 8.3% B2+, 45.3% transferrin receptor+, and 58.7% HLR-DR+. By adding each factor to both B cell fractions, we also demonstrated functional differences in the two populations. B cells in the earlier stage of activation only differentiated in response to IL 2 or IL 2 + IFN-gamma but not to BCDF, which was in contrast to B cells in the later stage that did not differentiate in response to IL 2 but did differentiate to BCDF. However, B cells in both stages proliferated in response to IL 2 but not to BCDF. Finally, we separated B cells in the later stage into two populations by the Percoll discontinuous gradient centrifugation. Lower density (larger) B cells were observed to proliferate but not to differentiate in response to IL 2, whereas higher density (smaller) B cells were observed to differentiate in response to BCDF. Therefore, we conclude that activated B cells initially become large and gain Tac-Ag and differentiate in response to IL 2 alone as well as the combination of IL 2 and IFN-gamma, whereas later in the more mature stage they become smaller again and differentiate into Ig-secreting cells only in response to BCDF.