Responses

To assess the functional integrity of our B cells, we measured ligand-induced changes in intracellular concentrations of free Ca²⁺ and cyclic adenosine monophosphate (AMP) and phosphorylation of specific sites within a panel of signaling proteins.

Ligand-Induced Changes in Intracellular Free [Ca2+]. Ca²⁺ responses caused by polyclonal goat anti-IgM antibody (0.3 mM), SDF-1 (6 nM), or LPA (1 mM) were evaluated using the calcium-sensitive dye Fluo-3

. Treatment of cells with these ligands induced a rapid and reproducible increase of intracellular [Ca²⁺] as shown in Fig 9. Three replicate tracings from one experiment are shown for each ligand. Similar tracings were obtained in additional experiments performed five or more times. Anti-IgM produced the greatest increase, which peaked at approximately 60 seconds, followed by a decrease to a plateau at approximately 20% of the peak value. SDF-1 and LPA produced tracings of smaller magnitude but with similarly reproducible increases in [Ca²⁺].

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Fig. 9. Ligand-induced changes in intracellular free [Ca²⁺]. Splenic B cells were incubated with 4 mM Fluo-3 AM at 5 x 10⁶ cells/ml and then adjusted to 7.5 x 10⁶ cells/ml in Hank's Balanced Salt Solution (HBSS) containing bovine serum albumin (BSA) for the assay. Anti-IgM (0.3 mM), SDF-1 (6 nM), LPA (1 mM), or vehicle was added at time 0. The assay was performed in a 96-well plate using a Fluoroskan Ascent Microplate Fluorometer. Measurements were recorded for 10 min followed by sequential addition of NP-40 (0.5% final) and EGTA (45 mM final) to obtain fluorescence maximum and minimum values, respectively. Intracellular free [Ca²⁺] was calculated using the equation: [free Ca²⁺] = K_d (F - F_min)/(F_max - F). The K_d for Fluo-3 was assumed to be 390 nM. Data was corrected for baseline drift caused by leakage of Fluo-3 AM from cells into Ca²⁺-containing medium. Each graph shows tracings of three replicate samples from one experiment. Experiments were performed at least five times.

Changes in Cyclic AMP Induced by Terbutaline or Prostaglandin E₂. Concentrations of cyclic AMP in B cells were determined by enzyme-linked immunosorbent assay (ELISA)

. Exposure of cells to the b2-adrenergic agonist terbutaline increases intracellular concentrations of cyclic AMP by activating the b2-adrenergic receptor, which in turn activates the heterotrimeric G protein G_s and the enzyme adenylyl cyclase. Prostaglandin E₂ (PGE₂) similarly activates the EP₂ and EP₄ receptors to increase cyclic-AMP concentrations. Effects of terbutaline and PGE₂ on cyclic AMP in isolated B cells were determined after exposure for 0.5, 1, 3, 8, and 20 minutes and in the absence of a cyclic nucleotide phosphodiesterase inhibitor (Fig. 10). A rapid, transient increase in cyclic AMP was observed that peaked at 0.5 minutes, followed by a sharp decrease to a sustained level approximately 40% of the peak response. Conversely, when cells were stimulated with PGE2, a rapid and relatively well-sustained increase in cyclic AMP was observed over the 20-minute period of observation.

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Fig. 10. Changes in total cyclic AMP induced by terbutaline or prostaglandin E2. B cells were incubated in 96-well plates at a concentration of 1.67 x 10⁷ cells/ml and treated with 10 mM terbutaline, 10mM prostaglandin E₂ (PGE2), or control buffer. Lysis buffer was added directly to the wells after 0.5, 1, 3, 8, or 20 min to stop the reaction. The amount of cyclic AMP in each sample is expressed as fmol/10⁶ cells. Each bar represents the mean ± SEM of samples obtained from five experiments.

Phosphorylation of Signaling Proteins. Western blot analysis

was used to measure changes in the phosphorylation of Akt (Ser473), p90RSK (Thr381), Stat 6 (Tyr641), and Erk1 and Erk2 (Thr183/Tyr185) in splenic B cells following stimulation with anti-IgM antibody, interleukin-4 (IL-4), or anti-CD40 antibody (Fig. 11). Stimulation with anti-IgM caused a rapid, increased phosphorylation of all proteins examined, except Stat 6. Maximal values were observed at 2.5 minutes, with Erk1 and Erk2 exhibiting the greatest increase. Anti-CD40 caused changes that mimicked those of anti-IgM, but the response was delayed until about 15 minutes after addition of ligand. By contrast, the response to IL-4 was characterized by a robust and sustained increase in the phosphorylation of Stat 6 (approximately 50-fold), while showing little or no effect on the other proteins. Phosphorylation of Akt followed a similar pattern of activation, but the increase was only 2.5-fold above that of untreated controls. These responses were reproduced in five independent experiments using different B-cell preparations.

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Fig. 11. Specific changes in protein phosphorylation induced by anti-IgM, interleukin 4 (IL-4), or anti-CD40. B cells were exposed to anti-IgM antibody (0.3 mM), IL-4 (0.34 nM), or anti-CD40 antibody (65 nM) for the indicated times. Cell extracts were analyzed by Western immunoblotting for changes in phosphorylation, first with a mixture of phosphospecific antibodies to Stat 6 (Tyr641), p90RSK (Thr381), Akt (Ser473), and Erk1 and Erk2 (Thr183/Tyr185). Following the initial processing, the blots were incubated with an antibody reactive to G protein b subunits 1 through 4 to correct for lane-to-lane variability in protein concentration. A representative blot is shown in panel (A). A positive control (+ Control) was prepared for use on all gels by exposure of a large number of cells to anti-IgM, IL-4, and protein phosphatase inhibitors. In panels (B-D) the quantification of Western blot signals are shown as the average + SD for five independent experiments performed with separate preparations of B cells. The same data were employed to calculate results as percent of positive control (bar graphs at right of each panel) or fold change (relative to the average for the samples of untreated cells; bar graphs at left of each panel).