Interleukin-3-treated non-B, non-T cells switch activated B cells to IgG1/IgE synthesis.

The switch of activated B cells to IgE synthesis is an interleukin (IL)-3-dependent process. It is currently thought that specific T cells activated by antigen presented in the context of class II major histocompatibility complex are the major source of IL-4. Recently it has been demonstrated that a splenic non-T non-B cell population (termed NBNT) has the capacity to produce IL-4 following IgE and IgG receptor cross-linkage. In this study we demonstrate that IL-4 producing NBNT cells can induce the switch of lipopolysaccharide-activated B cells to the synthesis of IgG1 and IgE antibodies. Furthermore, it was found that not only IgE receptor cross-linkage but IL-3 was able to stimulate NBNT cells to produce IL-4 and induce the switch of B cells to IgE synthesis. NBNT cells derived from the spleen and bone marrow of SCID mice were able to produce IL-4 on exposure to IL-3. This suggested that the ability of IL-3 to stimulate IL-4 production was not dependent on prior exposure of the NBNT cells to antibody complexes in vivo. Taken together these findings represent the first observation that enough IL-4 is produced by NBNT cells to actually influence a B cell IgG/Ig response. The findings also clearly demonstrate that B cells do not need high concentrations of IL-4 to be directed to switch to IgG1 and IgE synthesis.

Monoclonal antibodies that block adhesion of B cell progenitors to bone marrow stroma in vitro prevent B cell differentiation in vivo.

B cell differentiation requires adhesion of B cell progenitors to bone marrow (BM) or fetal liver stroma. We show that B lymphoid cells can adhere to the BM stroma cell line CS 1.3, in vitro. Two monoclonal antibodies, SAB-1 and SAB-2, inhibited the adhesion of a B220+ progenitor B cell line but did not interfere with the binding of cytoplasmic mu chain-positive pre-B cells or mature B cells to the BM stromal cell line. Injection of both SAB-1 and SAB-2 antibodies into pregnant mice reduced by 90% the number of B220+n B lineage cells in the livers of their embryos. Livers from such embryos also were virtually devoid of cells able to give rise to B cell colonies in soft agar cultures (CFU-preB). Either antibody separately had no effect. Flow cytometry analysis show that SAB-1 is present on CS 1.3 stroma cells and on a pre-B cell line while SAB-2 is present on pro-B and pre-B cell lines, but not on CS 1.3 stromal cells. SAB-1 and SAB-2 react with different molecules and neither antibody seems to recognize CD44, and adhesion molecule that may also participate in B cell differentiation. Proteinase K and trypsin can digest both SAB-1 and SAB-2 antigens from viable cells suggesting that both are cell surface proteins. We propose that antibodies SAB-1 and SAB-2 probably recognize novel cell-cell adhesion molecules, and that these molecules are involved in the interactions between B cell progenitors and stroma cells.

A sensitive and efficient induction system for murine IgE. Single cell analysis at the clonal level.

A culture system is described which permits the analysis of IgE expression by single murine cells within clones of B cells. The system is based on the use of a CB5.1 stroma cell line as a feeder which optimally supports the IL-4-induced switch to IgE of LPS-stimulated B cells in culture. In this system 100 U/ml IL-4 induces the switch to IgE, in 3-5% of B cells and the switch frequency to IgG1 was as high as 2%. Five ng IgE or 12 ng IgG1 were produced per clone containing on average 13-15 PFC. The detection of single IgE secreting B cells was possible due to two newly developed, highly specific rat anti-mouse IgE antibodies used in a sandwich-ELISA. The frequency of IgE-secreting B cells was enhanced 2.5 times when the fibroblastoid CB5.1 cells rather than thymocytes were used as feeder cells. CB5.1 cells supported the differentiation of B cells to IgM-PFC almost as well as rat thymocytes (which have, to date, been used as the standard feeder layer) whereas the amount of secreted IgG1 was about 3 times lower than in thymocyte cultures. Optimal switching to IgE occurred at concentrations of IL-4 which were 10-fold lower than that required for IgG1 expression, a situation quite opposite to that observed in the rat thymocyte-supported culture system. In confirmation of established data the switch of B cells to IgE or IgG1 occurred randomly. The advantages of CB5.1 cells as feeder cells are (1) the use of a homogeneous and defined cell line, (2) their limited release of defined lymphokines (IL-6 and GM-CSF), and (3) the low degradation and consumption of cytokine factors. The combination of the CB5.1 cell line with a highly specific IgE ELISA assay made it possible to analyse the appearance of IgE producing cells within a developing B cell clone.

Functional maturation of murine B lymphocyte precursors--III. Soluble factors involved in the regulation of growth and differentiation.

When 5-fluorouracil (5-FU) resistant bone marrow (BM) cells are depleted of B-cells and then cultured in insert chambers [separated from a layer of adherent BM (aBM) cells by a nucleopore membrane], no mature, lipopolysaccharide (LPS) reactive B-cells are formed. Factors acting on B-cell precursors are not produced unless nonadherent accessory cells have been cultured with aBM cells in the surrounding well. Moreover, soluble products are insufficient to induce differentiation of B-cell precursors unless the cells have been conditioned by direct contact with aBM cells. Such preconditioned precursors complete differentiation when cultured with IL-3 plus IL-1 in dishes coated with fibronectin. In cultures supplemented with IL-3, IL-1 and fibronectin, a pleomorphic layer of aBM cells is generated after a few days. This is not the case in cultures lacking IL-3. Therefore, an important function of IL-3 may be to recruit an adherent accessory cell type from the pool containing precursors of the B-cell as well as myeloid lineages. This view is further supported by experiments on the generation of colonies containing antibody secreting B-cells from day 15 fetal liver precursors which depends on soluble products secreted by aBM cells. When aBM cells established in the absence of IL-3 are present, more than one cell type (or cell product) is limiting. However, if aBM cell layers are generated in the presence of IL-3, only B-cell precursors seem to be limiting. Since macrophages play an important role in the aBM population, the effect of CSF-1 was investigated. Even though CSF-1 potentiates the effect of IL-3 and IL-1, it cannot replace these interleukins. Like IL-3, it may influence B-cell differentiation in an indirect manner by modifying the microenvironment. Another important function of macrophages seems to be related to the production of C3, which binds to CR2 after degradation. P14, a peptide of the CR2 binding C3d fragment, strongly inhibits maturation of B-cell progenitors. A larger CR2 binding peptide, P28, is inhibitory at low concn but stimulatory at higher concn. It is assumed that aggregated P28 may cross-link with CR2 and thereby transfer a differentiation signal to the cell.

[Development of the glucocorticoid secretion of the adrenal glands of normal chick embryo and after late decapitation]. / Evolution de la sécrétion en glucocorticoïdes de la surrénale chez l'embryon de poulet normal et après décapitation tardive.

The competitive protein-binding assay of glucocorticoids in Chick embryo shows a significant increase of these adrenal hormones in plasma by day 13 of incubation. From the effects of the hypothalamo-hypophysis axis removal by late decapitation, we may conclude an hypophyseal control on the adrenal gland on the 13th day. The following decrease of plasmatic hormone concentration might be due to a negative feed-back. The pituitary hormonal control appears again by day 19 of incubation.