Interleukin 2 (IL-2) stimulated activation of the 42-kD extracellular signal-regulated kinase 2 (Erk2) in murine IL-3-dependent cells, expressing either high or intermediate affinity IL-2 receptors. Activation was both rapid, occurring within 5 min of IL-2 addition, and prolonged, remaining elevated for 30 min. Activation of Erk2 appeared to be necessary for IL-2 stimulation of proliferation, as deletion of a region of the cytoplasmic domain of the IL-2 receptor beta chain, essential for IL-2 stimulation of proliferation, abolished Erk2 activation by IL-2. Furthermore, cells that had been deprived of cytokine for 24 h were then refractory to IL-2 stimulation of both Erk2 activity and proliferation. However, elevation of Erk2 activity was not sufficient to stimulate proliferation, as protein kinase C activation stimulated Erk2 activity but not DNA synthesis. Also, cells exposed to IL-2 in the presence of rapamycin showed full Erk2 activation but not DNA synthesis. These data suggest that IL-2 must stimulate both Erk2 activity and a further pathway(s) to trigger cell proliferation.
Murine bone marrow-derived cells, dependent on interleukin 3 (IL-3) for their growth in culture, undergo programmed cell, or apoptosis, upon cytokine withdrawal. Here it is reported that a variety of DNA damaging agents cause a more rapid onset of apoptosis in a factor-dependent cell line, BAF3, deprived of IL-3. In contrast, when cultured in the presence of IL-3, or other growth promoting factors, BAF3 cells are highly resistant to X-irradiation and the cytotoxic drugs etoposide and cisplatin. Overexpression of the bcl2 gene product also protects BAF3 cells from DNA damage. The presence of IL-3 is not required during the initial events of DNA damage or its repair. In the absence of IL-3, cells still complete the repair of DNA breaks within 15 min, and continue to cycle for 5 h. At this time, IL-3 is necessary to prevent the accelerated onset of DNA cleavage from a G2 arrest point.
We have shown that, by suitable idiotypic manipulation, BALB/c mice can express the major cross-reactive idiotype (CRI) of A/J mice in response to azophenylarsonate (Ars). In order to know if the CRIA idiotype is present in the potential repertoire of BALB/c before any intentional selection, we used polyclonal activation in vitro and limiting dilution analysis. The readout was done with two monoclonal anti-CRIA antibodies that recognize distinct idiotopes on a CRIA+ A/J germline-encoded monoclonal antibody. We studied the frequency of CRIA+ lipopolysaccharide (LPS)-reactive cells in the spleens of nonimmune and immune A/J mice and in the spleens of naive and manipulated (i.e., producing CRIA+ antibodies) BALB/c mice. A/J and BALB/c naive individuals presented very high frequencies of Ars-specific B cells while the frequency of CRIA+ B cells was only a minor subset (0.5%) of the total Ars-specific subset in the two strains. When A/J mice were immunized with Ars-keyhole limpet hemocyanin, a clear preferential expansion of the CRIA+ minor subset of A/J mice was observed (100x). No such enhancement was observed in BALB/c mice similarly treated. Manipulated BALB/c mice presented a higher frequency of CRIA+ anti-Ars B cells than naive or antigen-immunized BALB/c individuals.