Interleukin-2 inhibits the interleukin-4-induced human IgE and IgG4 secretion in vivo.

The effect of interleukin-2 (IL-2) on IL-4-induced IgE and IgG4 secretion by B cells in peripheral blood mononuclear cell (PBMC) preparations from non-atopic healthy humans and atopic dermatitis patients was investigated. PBMC were cultured at an optimal concentration of recombinant IL-4 with or without addition of IL-2 for 10 days. Native and recombinant IL-2 inhibited the IL-4-induced IgE and IgG4 secretion in a dose-dependent manner by cells from both normal and atopic donors. Rabbit antibodies to IL-2 or to the monoclonal anti-IL-2 receptor antibody anti-TAC reversed the IL-2 effect. Culturing cells with IL-4 and IL-2 for 1 or 2 days only slightly suppressed the IgE and IgG4 secretion whereas addition of IL-2 to IL-4 containing cultures on day 4 or 5 inhibited the IgE and IgG4 secretion more effectively. This is in contrast to interferon-gamma (IFN-gamma) which inhibited the IL-4 induced IgE and IgG4 secretion when added for the first 24 or 48 h but had no effect when added on days 4 or 5. The data demonstrate that both IL-2 and IFN-gamma act as antagonists in the IL-4-induced IgE and IgG4 secretion by human B cells; while IL-2 appears to inhibit relatively late in culture, IFN-gamma has an early inhibitory effect, suggesting that the two lymphokines inhibit the IL-4 effect by different mechanisms.

Interleukin-4 induced IgE and IgG4 secretion by B cells from atopic dermatitis patients.

Peripheral blood mononuclear cells from 8 normals and 8 patients with atopic dermatitis (AD) were cultured with recombinant interleukin-4 (IL-4) and the IgE and IgG subclass levels in the culture supernatants measured by radioimmunoassays. IL-4 induced IgE and IgG4 secretion by B cells from both normals and AD patients whereas it has no consistent effect on IgG1, IgG2 and IgG3 secretion. The IL-4 dose response was similar for IgE and IgG4 secretion by cells from both normals and AD patients. On the average, the patients' cells secreted more IgE and less IgG4 than the cells from normals, but because of a large variation, the differences were not significant. However, the ratio of IgG4:IgE secretion was significantly greater for normals than AD patients (mean +/- SEM 7.1 +/- 1.6:1 vs. 1.5 +/- 0.4:1; p less than 0.01). The data demonstrate that IL-4 induces IgE and IgG4 secretion by B cells from both normals and AD patients and suggest that the IL-4 induced switch from IgM to IgG4 or IgE secretion may proceed preferentially to IgE in AD patients as compared to normals.

Effects of in vitro corticosteroids on B cell activation, proliferation, and differentiation.

The present study demonstrates the graded effect of in vitro corticosteroids (CSs) on the different phases of B cell activation, proliferation, and differentiation. Early events such as activation and proliferation of high-dose anti-mu or Staphylococcus aureus-stimulated B cells are profoundly suppressed by the presence of in vitro CSs. The suppressed proliferative response may be mediated by a direct effect on B cells and/or modulation of accessory cell function. Later events in the B cell cycle such as the proliferative response to B cell growth factor after either in vivo or in vitro activation are less sensitive to the suppressive effects of in vitro CSs. The final events in the B cell cycle; namely, the differentiation to the immunoglobulin-producing state, is not suppressed by in vitro CSs. Indeed, depending on the systems employed, there is either no effect or enhancement of immunoglobulin secretion by the presence of in vitro CSs. The graded effect of in vitro CSs on the discrete phases of the B cell activation, proliferation, and differentiation cycle provide new insights into the complex nature of CS-induced modulation of human B cell responses.

Direct effects of a monoclonal B cell differentiation factor and of purified interleukin 1 on B cell differentiation.

We have previously described the presence of factors in mixed lymphocyte culture supernatants that induce activated but not resting human B cells to secrete Ig. In the present study, we describe the effects of a B cell differentiation factor in the supernatant (SN) of a T-T hybridoma (clone 7D5). We find that it induces Ig secretion by human B cells in the absence of T cells or monocytes. It acts only on activated B cells, because small (resting) B cells isolated by centrifugal counterflow elutriation do not respond to it, whereas the same cells do develop into Ig-secreting cells if activated in culture with Staphylococcus aureus Cowan I before exposure to SN 7D5. By using class-specific reagents in enzyme-linked immunosorbent assays, we found SN 7D5 to result in the secretion of significant amounts of IgM, IgG, and IgA. We also studied the effects of highly purified interleukin 1 on this differentiation process. Interleukin 1 by itself failed to induce Ig secretion by activated B cells, and its presence was not required for the induction of Ig secretion by SN 7D5. However, interleukin 1 consistently synergized with SN 7D5 in inducing Ig secretion by purified B cells.

Development of a human T-cell hybridoma secreting separate B-cell growth and differentiation factors.

A cloned human T-cell hybridoma (7D5) secreting B-cell growth factor (BCGF) and B-cell differentiation factor (BCDF) was established. Supernatant from this hybrid was capable of maintaining proliferation in anti-IgM-activated normal human B cells. In addition, the hybridoma supernatant induced differentiation and antibody secretion in Staphylococcus aureus Cowan I-stimulated B cells. No interleukin 2 was present in supernatant from this hybridoma. Molecular size of the hybridoma-derived BCGF and BCDF was determined by gel filtration chromatography. BCGF activity was present in the 20-kDa fractions, and BCDF activity eluted in the 30- to 35-kDa fractions. The isoelectric points of the factors, determined by chromatofocusing, were 6.6 for BCGF and 5.9 for BCDF. Finally, absorption experiments were performed using specific target cells. Phytohemagglutinin-stimulated T-cell blasts did not remove either BCGF or BCDF activity. Anti-IgM-activated B cells absorbed BCGF but not BCDF. In contrast, CESS cells removed BCDF but not BCGF. Thus, a human T-cell hybridoma secreting two distinct B-cell lymphokines was developed. Further immunochemical and functional studies of these immunoregulatory molecules should greatly enhance our understanding of the regulation of human B-cell function in normal and disease states.

Activation and immunoregulation of human B lymphocytes.

Immunocompetent cells communicate via direct cellular contact and/or by the release and binding of soluble mediators. These soluble mediators transmit signals for growth and differentiation of various cell types. We have been intensively studying the regulation of human B lymphocytes and have focused on the events which occur following stimulation of mature, resting B cells with antigen or an antigen equivalent, anti-immunoglobulin antibody. Anti-Ig stimulates resting B lymphocytes to enlarge, synthesize RNA, increase membrane Ia expression, express activation markers, and become responsive to soluble factors termed B-cell growth factors (BCGF). We have described two different BCGFs, an 18 kd BCGF derived from a T-T hybridoma and a 60 kd BCGF derived from a T cell line. Activated B cells in the presence of BCGF further enlarge; express another activation marker, the transferrin receptor; and enter the S phase of the cell cycle, but do not differentiate unless another factor is present, e.g., B-cell differentiation factor (BCDF). We have described another T-T hybridoma which constitutively secretes both an 18 kd BCGF and a 35 kd BCDF. These two factors can easily be separated by biochemical means. The 35 kd BCDF induces the differentiation of activated but not resting B cells. Besides these B-cell-specific factors, we have studied the immunoregulatory effects of interleukin 1 (IL-1), IL-2, and interferons (alpha and gamma) on human B-cell responses. Interleukin 1 weakly co-stimulates resting B cells when it is present with anti-Ig and enhances the differentiation of activated and proliferating B cells when it is present in culture with BCDF. Interleukin 2 receptors as defined by the monoclonal antibody anti-Tac and radiolabeled IL-2-binding assays are present on in vitro activated B cells. Recombinant IL-2 added to cultures of in vitro activated B cells promotes both B-cell growth and B-cell differentiation into Ig-secreting cells. Finally, interferons appear to have little direct effect on human B-cell function. Major advances in our understanding of the complexities of B-cell activation, proliferation, and differentiation have been realized over the past few years. The eventual isolation and chemical characterization of the soluble mediators of B-cell function and the receptors for these mediators should lead to further insights and to new approaches to those diseases characterized by aberrations of B-cell function.

Selective suppression of an early step in human B cell activation by cyclosporin A.

The effect of cyclosporin A (CsA), a fungal metabolite with immunosuppressive properties, on the induction of human B cell proliferation and differentiation, has been described. CsA had a selective inhibitory effect on the activation phase of the cell cycle vs. the proliferation phase following preactivation of the cells. Cell enlargement and RNA synthesis of small resting B cells triggered by anti-mu were inhibited by addition of CsA (5-500 ng/ml). The inhibitory effect of CsA was found only when the drug was added within 24 h of initiation of culture. In marked contrast, once small B cells were activated by anti-mu, the resulting large, activated B cells could be induced to initiate DNA synthesis by incubation with B cell growth factor (BCGF), and addition of CsA (1-1,000 ng/ml) to the culture did not suppress this BCGF-induced B cell proliferation. Addition of CsA to cultures of B cells which had been preactivated with Staphylococcus aureus Cowan strain I (SAC) and were already proliferating did not suppress B cell differentiation factor (BCDF)-induced differentiation of these cells. Thus, these data indicate that CsA can be used as a pharmacologic tool to dissect out human B cell responses into two distinct steps: (a) the initial activation step induced by anti-Ig, which is characterized by cell enlargement, RNA synthesis, and expression of receptors for BCGF; and (b) the proliferative step induced by BCGF in these preactivated B cells that undergo DNA synthesis and can then go on to differentiate in the presence of BCDF. In this regard, CsA selectively suppresses an early step of human B cell activation and has little inhibitory effect on the subsequent factor-dependent proliferation and differentiation.

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.

The relationship between immunization and circulating antigen-specific plaque-forming cells.

The relationship between the appearance of cells producing antibody to tetanus toxoid (TT) in the circulation and the serum titers of anti-TT IgG following booster immunization has been studied. It was found that cells producing anti-TT antibody can be detected in the circulation in a hemolytic plaque assay using sheep red blood cells (SRBC) coated with TT by the chromic chloride method. In symmetric inhibition studies using cells from TT or keyhole limpet hemocyanin (KLH)-immune donors, the homologous antigen inhibited 100% of the PFC with no cross-inhibition. Thus, the plaque-forming cells (PFC) detected in this assay are specific for the immunizing antigen. No evidence of polyclonal B-cell activation in response to TT was found, as shown by a failure to detect any PFC against unmodified or KLH or human serum albumin-treated SRBC. In addition, the increase in total Ig-secreting cells observed in a staphylococcal protein A reverse hemolytic plaque assay was always accounted for by the number of anti-TT antibody-producing cells observed. The peak number of anti-TT antibody-producing cells varied between donors, but the kinetics of their appearance was highly reproducible--none before Day 5, peak numbers between Days 6 and 8, and a sharp decline with only rare anti-TT Ig-secreting cells in the circulation by Day 15 postimmunization. Anti-TT antibody-producing cells appeared in the circulation prior to any detectable increase in serum anti-TT antibody titers, and following the disappearance of PFC from the circulation, there was no further increase in serum IgG anti-TT levels. These observations demonstrate a marked specificity of B-cell activation on boosting with a recall antigen, and a parallelism between the appearance of activated B cells in the circulation and of IgG anti-TT synthesis by the subject as a whole.