Essential role of membrane cholesterol in accelerated BCR internalization and uncoupling from NF-kappa B in B cell clonal anergy

Abstract

Divergent hypotheses exist to explain how signaling by the B cell receptor (BCR) is initiated after antigen binding and how it is qualitatively altered in anergic B cells to selectively uncouple from nuclear factor kappaB and c-Jun N-terminal kinase pathways while continuing to activate extracellular signal-regulated kinase and calcium-nuclear factor of activated T cell pathways. Here we find that BCRs on anergic cells are endocytosed at a very enhanced rate upon binding antigen, resulting in a large steady-state pool of intracellularly sequestered receptors that appear to be continuously cycling between surface and intracellular compartments. This endocytic mechanism is exquisitely sensitive to the lowering of plasma membrane cholesterol by methyl-beta-cyclodextrin, and, when blocked in this way, the sequestered BCRs return to the cell surface and RelA nuclear accumulation is stimulated. In contrast, when plasma membrane cholesterol is lowered and GM1 sphingolipid markers of membrane rafts are depleted in naive B cells, this does not diminish BCR signaling to calcium or RelA. These results provide a possible explanation for the signaling changes in clonal anergy and indicate that a chief function of membrane cholesterol in B cells is not to initiate BCR signaling, but instead to terminate a subset of signals by rapid receptor internalization.

Figure 1.

Anergic B cells fail to accumulate RelA in the nucleus but contain a large pool of intracellular IgD. (A) Immunofluorescent staining and confocal observations were performed on Triton X-100–permeabilized naive B cells (BCR tg) or anergic B cells (BCR tg x sHEL tg). Staining for IgD^a and RelA was analyzed after HEL stimulation (HEL 30 min), goat anti-κ light chain stimulation (Anti-Kappa 30 min), or without stimulation (Unstim.). Note that the thin rim of cytoplasmic and plasma membrane staining for IgD readily demarcates the nucleus of these small lymphocytes. (B) IgD staining was performed on Triton X-100–permeabilized or on nonpermeabilized (no TX100) cells to compare the distribution of surface receptors only with the distribution of intracellular plus surface IgD receptors. (C) 300–900 permeabilized cells were observed per condition per experiment, and cells displaying a polarized accumulation of IgD^a at one pole in the focal plane were counted. Mean + SD values are obtained from three to six independent experiments. (D) As in C, except that cells were either not permeabilized (no TX100) or permeabilized (TX100) before staining for IgD. (E and F) As C and D, cells displaying predominantly nuclear localization of RelA in the midline focal plane of the cells, as shown in A, were counted. Open bars, BCR tg; filled bars, BCR tg x sHEL tg. Stimulation time is 30 min for C, D, and E and as indicated in F.

Figure 2.

Polarized intracellular accumulation of IgD and antigen in anergic B cells. (A) Representative confocal images of naive or anergic B cells. Cells were stimulated as indicated, placed on ice, stained with saturating doses of an anti–IgD^a-FITC mAb (IgD^a-FITC, surface), and then fixed, permeabilized, and stained with an anti-IgD^a mAb-biotin and SAV-Cy3 (IgD^a-Cy3, permeabilized) to reveal intracellular IgD. (B) FACS staining on unstimulated naive or anergic cells, either nonpermeabilized (bold lines) or saponin permeabilized (shaded histograms). Surface BCRs available to bind HEL were measured by staining with HEL-biotin before saponin treatment, followed by streptavidin-PE, whereas all BCR-bound HEL antigen was stained with anti–HEL cychrome antibody. Note that the anti-HEL cychrome staining of anergic cells is twice as high on permeabilized cells compared with nonpermeabilized cells, demonstrating that half of the HEL-engaged BCRs are located intracellularly. There is no difference in surface IgD staining between the naive and anergic cells, and the lower staining for HEL-biotin on the latter reflects occupancy of half of the surface BCRs with untagged HEL and down-regulation of surface IgM.

Figure 3.

Effects of depleting cholesterol from membranes of naive and anergic B cells. (A) Surface levels of raft-associated GM1 sphingolipids and IgD^a were measured by flow cytometry before (gray histogram) and after treatment with the indicated concentrations of MBCD (open histograms). The dotted lines serve as an arbitrary visual reference for control fluorescence levels. (B) Membrane cholesterol levels were measured by staining with filipin and flow cytometric measurement of filipin fluorescence on untreated cells (Control), MBCD-treated cells (MBCD), or cells treated with MBCD that had been preloaded with cholesterol (MBCD-Chol). (C) Values of the mean fluorescence of the filipin staining were normalized with the value obtained for untreated and unstimulated BCR tg cells. Open bars, BCR tg cells; filled bars, BCR tg x HEL tg cells. Data shown are representative of two to five independent experiments. (D) HEL antigen-induced calcium responses were recorded as variations in the Indo-1 fluorescence ratio gated on the B220⁺ cell population. Bold lines, calcium response of control cells; thin lines, cells that were first pretreated with 2.5 mM MBCD. Stimulation is performed after 60 s with soluble HEL. Experiment shown is representative of five independent experiments.

Figure 4.

MBCD treatment restores RelA nuclear accumulation in anergic B lymphocytes. (A) Staining for IgD^a and RelA and confocal analysis were performed on permeabilized B cells. The cells were first treated with MBCD or left untreated, and then either stimulated with HEL for 30 min or left unstimulated, as indicated. (B) Quantitation of cells with nuclear RelA accumulation. Open bars, naive BCR tg cells; filled bars, anergic BCR tg x HEL tg cells. Mean + SD is calculated from three independent experiments, where 300–900 cells were counted each time. (C) As in B, but cells with polarized IgD^a staining were counted.

Figure 5.

Enhanced BCR endocytosis in anergic cells. (A) Histograms showing HEL-PE fluorescence of B220⁺ naive (BCR) or anergic (BCR tg x sHEL tg) cells. Cells were either acid treated to inactivate the fluorescence of cell surface PE so remaining signal measures endocytosed antigen (shaded histograms), whereas control cells (open histograms) provide a measure of total surface and intracellular HEL-PE. Cells kept at 4°C serve as a no endocytosis control, whereas cells warmed to 37°C for 10 min before acid treatment reveal the fraction of bound antigen that has been internalized. (B) Time course of antigen endocytosis. •, anergic B cells; ◯, naive B cells. Dashed lines show endocytosis by cells with cholesterol lowered by 2.5 mM MBCD, and solid lines are corresponding values for mock-treated controls. The experiment shown is representative of three independent experiments. The right panel shows the rate of endocytosis for the first 10 min calculated from the dashed line on the left panel. Values represent the mean of three independent experiments. (C) Pharmacological inhibition of antigen endocytosis in naive cells (open columns) and anergic cells (filled columns). The amount of antigen endocytosed by untreated cells after 10 min was set to 100%. The effect of each treatment is expressed as relative endocytosis compared with the untreated value (Untd.). PP1, rottlerin (Rottl.), and latrunculin B (Lat B) were used at the indicated concentrations. Data shown are representative of two to five independent experiments.

Figure 6.

Rapid recycling of antigen-loaded BCRs to the cell surface. (A) Naive or anergic B cells were incubated with HEL-SS-biotin at 4°C, warmed to 37°C for 10 min to allow endocytosis, and then the remaining surface antigen was cleaved of biotin with MESNA. The cells were then warmed again to 37°C for the indicated times and stained with streptavidin-APC to detect material returning to the cell surface. The experiment shown is representative of three independent experiments. (B) Percentage of reexpressed receptor bound to antigen in three independent experiments.