Role and regulation of interleukin ( IL )-2 receptor a and 13 chains in IL-2-driven B-cell growth

Substantial proportions of resting B cells constitutively express low levels of IL-2 receptor (IL-2R) a and/or . 3 chains. The expression ofthese chains is differentially regulated by anti-IgM and IL-2/IL4. The anti-IgM induces IL-2R a chain expression, whereas each of the two cytokines induces IL-2R (3 chain expression in a dose-dependent manner. Moreover, IL-2 induces the growth of B cells, when the cells were pretreated with IL-2 or IL-4 for 24 h. The magnitude of this IL-2-driven B-cell growth depends upon the level of IL-2R (3 chain expression. Costimulation of the B cells with IL-2 and anti-IgM shifts the dose-response curve, and the cells proliferate at an IL-2 concentration as low as 40 pM. These results indicate that the levels of anti-IgM-induced IL-2R a chain and IL-2-induced IL-2R (3 chain determine the sensitivity of the cells to IL-2. The high-affinity IL-2 receptor (IL-2R) on the cell surface of lymphocytes consists of at least two IL-2-binding molecules, IL-2R a chain (IL-2Ra or p55) and IL-2R f chain (IL-2RP or p70-75) (1-8). IL-2Rf3 has an intermediate IL-2-binding affinity (1-8). Cloning and expression studies of the cDNA for human and murine IL-2Rj3s have revealed that this IL-2binding protein contains a large cytoplasmic region essential for ligand-mediated intracellular signal transduction (9-11). In contrast, IL-2Ra has a low IL-2-binding affinity and contains a relatively short cytoplasmic region (12-14). Although the IL-2Ra is not directly responsible for the intracellular IL-2 signal transduction (12-14), it is essential for the formation of the high-affinity IL-2R in conjunction with IL-2Rf3 (15, 16). Thus elucidation of the regulatory mechanism for expression of these receptors and definition of the relative role of each receptor in IL-2 response are essential for understanding the regulation of lymphocyte growth. There is much evidence that IL-2 plays a role in B-cell growth and differentiation (17-24). In fact, several stimuli capable of inducing the expression of IL-2Ra on B cells have been reported (17-20, 25), and recently, stimuli that induce IL-2RP3 on B cells have also been reported (20-22). Here we demonstrate the constitutive expression of IL-2Ra and P3 on resting B cells, their differential regulation by anti-IgM and IL-4/IL-2, and the IL-2-dependent growth of IL-4or IL-2pretreated B cells mediated by up-regulated IL-2Rfs. MATERIALS AND METHODS Reagents. Goat anti-IgM, rabbit anti-asialo-GM1 (Wako Pure Chemical, Osaka, Japan), and rat antibodies, 7D4 (IgM) and 3C7 [IgG; specific for IL-2Ra (25, 26)], TM-P1 [IgG2b; specific for IL-2Rl3 (27)], and Bet-II (IgG anti-IgM) were used. As controls for 7D4, 3C7, and TM-P1, we used 49C2D6R5 (49C2; IgM anti-trinitrophenyl), liB11 (IgG antiIL-4), and rat IgG2b myeloma, respectively. Preparation of B Cells. B cells were purified from 7to 9-week-old female BALB/c mice by Leibson's method (28), which is capable of depleting activated lymphocytes, macrophages, and T cells. However, resultant cells still retained a few percent of natural killer cells, which are highly IL-2responsive (29). Therefore, we depleted natural killer cells and employed two B-cell populations: (i) panned B cells (-99% surface IgM positive), obtained by sequential complement-mediated T-cell lysis (28) and positive selection on an anti-IgM-coated dish (30). This process did not activate B cells; (ii) B cells (-98% surface IgM positive), obtained by negative selection (28) from anti-asialo-GM1-pretreated mice (50 ILI per mouse, 3 days before use). Factors. We used molecularly cloned murine IL-4 and human IL-2. Activity of IL-4 was determined as described (31). Culture Conditions. B cells (3 x 106 cells per ml per well) were cultured alone or with anti-IgM (10 jig/ml), IL-4, and IL-2, either individually or in various combinations for various periods. Each group was set up with multiple wells. Flow Cytometric Analysis of IL-2Ra and IL-2R(8. B cells were first stained with fluorescein isothiocyanate-conjugated Bet-II or 7D4 and subsequently stained with a battery of biotin-conjugated TM-,81 and phycoerythrin-labeled avidin. Stained profiles were obtained from a FACScan analyzer. Preparation of CytoplasmicRNA and Dot Blot and RNA Blot Analyses. Preparation of cytoplasmic RNA and dot blot and RNA blot analyses were carried out as described (32). Yields of RNA from 107 resting B cells and 107 stimulated B cells were generally 10 jig and 25 pug, respectively. DNA Probes. We used pmIL-2R8-cDNA (14) and the Pvu II fragment (540 base pairs) of MIL2R/3-26 (10) to detect IL-2Ra mRNA and IL-2R/3 mRNA, respectively. A T-cell receptor probe (86TS) and probes for rRNA and P-actin mRNA were used to examine the purity ofB cells and to demonstrate equal RNA loading, respectively. Proliferation Assay. B cells (3 x 106 cells per ml per well), cultured for 24 h with medium alone or with IL-4 or IL-2 in the presence or absence of anti-IgM, were collected, washed, and recultured in flat-bottomed 96-well microtiter plates at 1 x 105 cells per well in the presence of serially diluted IL-2 with or without rat monoclonal antibodies for 3 days, with 1 jxCi (1 Ci = 37 GBq) of [3H]thymidine during the final 16 h.