Immunoglobulin heavy chain mRNA in mitogen‐stimulate B cells

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Abstract
This paper relates the synthesis of DNA, immunoglobulin and heavy chain (H) MRNA in murine spleen cells following activation of B cells with lipopolysaccharide from E. Coli (LPS). Spleen cells (CBA/H mice) were cultivated with 10 % FCS and 10 μg LPS/ml. 4 h pulses with [3H]thymidine showed that DNA synthesis was stimulated within the firstb day following LPS activation and exhibited a sharp peak at 24 h. The shape of the DNA synthesis curve suggests that the cells susceptible to LPS stimulation are activated in a synchronous manner. Stimulation of H‐chain mRNA (H‐mRNA) synthesis proceeded raidly (within 6 h of LPS addition) and peaked around 24 h, in parallel to DNA synthesis. The H‐mRNA was isolated and quantitated by making use of its interaction with IgG [1, 2]. The actual level of H‐mRNA in the culture increased three‐fold during the first 24 h and then doubled within the next 48 h. Estimates of the actual number of H‐mRNA were approximately 200 molecules H‐m‐RNA/cell on day 0 rising to 1800/cell on day 3. In such a mixed cell populations these figures will be accurate only within a factor of 2–3 (at least 35 % B cells in spleen cell suspensions at the commencement of the culture, with up to 35–60 % of plasma blasts by day 3 and 4 of LPS treatment). Translation of the lymphoid cell mRNA in oocytes from Xenopus laevis demonstrated that stimulation of H‐mRNA synthesis was restricted to μ‐mRNA, although some γ‐mRNA was present in the original spleen cells. High levels of synthesis of immunoglobulin followed after a lag period of about 24 h following LPS addition peaking after 48 h adn 72 h; the proportional Ig production relative to total protein synthesis reached 26 % on days 3 and 4. Stimulation of Ig production was limited to IgM. Rapid stimulation of mitosis and H‐mRNA synthesis thus precedes the maximum synthesis of Ig molecules, suggesting a translational block on the transport of H‐mRNA from the nucleus during early stages of activation.