Induction of the c-myc protooncogene after antigen binding to hapten-specific B cells.

Abstract

A central question in B cell biology concerns the potential role of the surface immunoglobulin (sIg) receptor for antigen as a means for signal transduction during the process of antigen-driven B cell activation . The original one-signal hypothesis (1) predicted that sIg functioned merely to focus antigen onto the surface of antigen-responsive cells. The validity of such a conclusion has been questioned by results from many studies (2, 3) in which anti-Ig reagents were used to successfully activate resting B cells. Most importantly, the crosslinking of sIg by such reagents has been shown to move resting (G o) B cells into at least the G, stage of the cell cycle (4). However, when populations of hapten-specific B cells were stimulated with thymus-dependent (TD) antigens, the antigen-binding cells did not leave Go (5, 6), even though they were shown to bind and cap antigen (6). Recently, we have readdressed (7) this issue by searching for biochemical consequences of antigen binding to the sIg receptor . In this regard, we have shown that TD antigenic stimulation of hapten-specific B cells generated a detectable phosphatidylinositol cycle . In the present communication, we have shown that such antigenic stimulation of hapten-specific B cells induced elevated levels of c-myc mRNA . These observations lend further support to the concept that antigen binding to the sIg receptor results in the transduction of signals to the cell and directly implicates the active participation of sIg during the process of antigen-mediated B cell activation . Materials and MethodsAnimals. DBA/2 female mice were obtained from Harlan Sprague-Dawley, Inc. (Indianapolis, IN) and were used at 8-10 wk of age.Preparation of TNP-ABC. The method used for the isolation of TNP-antigen-binding B cells (TNP-ABC) has been described elsewhere (8). The resultant cell preparations were 80-90% rosette forming cells (RFC).Culture Conditions . The TNP-ABC were cultured in flat-bottomed 24-well plates (Costar, Cambridge, MA) at 2 x 106 cells/well in 1 .0 ml of RPMI 1640 with penicillin, streptomycin, glutamine, 10% FCS (Sterile Systems, Logan, UT) and 50 AM 2-ME. Cultures were incubated in a humidified atmosphere of 83% N2, 10% C02, 7% 02 at 37°C . After the 12-18 h preculture period, the appropriate reagents were added in 10 Al of complete medium and the cells were cultured for an additional 2 h.Analysis of c-myc mRNA Level. Total cellular RNA was isolated as described by Glisin et al. (9). For the dot blot analysis, aliquots of the recovered RNA plus 10 jug of yeast tRNA were applied to a nitrocellulose filter using a dot blot Minifold (Schleicher and This work was supported by grant AG0476101 from the National Institutes of Health . 944J. Exp. MED .

Thymus‐Dependent Antigenic Stimulation of Hapten‐Specific B Lymphocytes

“…It was initially documented that TD antigenic challenge of hapten-specific B-cells results in a rapid depolarization of the plasma membrane (Monroe & Cambier 1983a). We (Grupp et al 1987) as well as others (Myers et al 1987) have shown that a similar challenge of TNP-ABC induces a detectable PI cycle and elevation in the level ot c-myc mRNA (Snow et al 1986). Membranes purified from PMA-stimulated B-cells demonstrate the appearance of at least 10 unique or hyperphosphorylated proteins ( Fig.…”

COOH terminus of membrane IgM is essential for an antigen-specific induction of some but not all early activation events in mature B cells.

“…One of the immediate reactions includes activation of phospholipase C-catalyzed hydrolysis of phosphatidyl inositol into inositol 3-phosphate and diacylglycerol, followed by a rise in intracellular Ca 2+ levels (4). One of the next documented consequences is induction of various immediate-early gene messages such as Egr-1, c-fos, Jun, c-myc (5)(6)(7). Recently, several laboratories have identified phosphorylation of proteins on tyrosine residues after crosslinking of mlgM (8)(9)(10), indirectly implicating the activation of tyrosine kinase(s).…”

Isolation and characterization of a B lymphocyte mutant with altered signal transduction through its antigen receptor.

“…Transformed B cell lines representative of specific maturation stages have proven useful for studies ofreceptor Ig signal transduction . For example, the murine B lymphoma WEHI-231 displays signal transduction events similar to normal B lymphocytes with respect to G protein-linked PI hydrolysis (2, 12), Cat+ mobilization (13)(14)(15), protein phosphorylation (16), and induction of cfos (8) and c-myc (17,18) expression. The coupling oflater events in WEHI-231 appears different from normal mature B lymphocytes, since sIg-generated signals are translated by WEHI-231 to cause decreased proliferation and eventually cell death (19,20).…”