Baruch Toledano

Intravenous immunoglobulin inhibits IgE production in human B lymphocytes

BACKGROUND Intravenous immunoglobulin (IVIG) is commonly used as both an immune-enhancing and immune-modulating agent. Treatment with high doses of IVIG diminishes IgE secretion in patients with severe steroid-dependent asthma. OBJECTIVE We studied the action of IVIG on IgE production in highly purified B lymphocytes stimulated without additional T cells to determine the action of IVIG on B lymphocytes. METHODS Human B cells were purified from tonsils, and T lymphocytes were removed by E-rosetting. B cells were cultured with IL-4 (400 U/mL) and anti-CD40 antibodies (1 microg/mL¿, with or without additional IVIG. Cell proliferation was determined by 3[H]-thymidine uptake, and supernatant IgE was determined by ELISA. Cell cycle analysis was performed by flow cytometry, and IgE transcripts were measured by in situ hybridization. RESULTS IVIG (5 mg/mL) decreased B-cell proliferation in IL4/anti-CD40-stimulated B cells by an average of 74% (+/-6%). Addition of IVIG up to 48 hours after initiation of cell culture led to significant diminution of cell proliferation at 96 to 120 hours. This effect was dose dependent, with 10 mg/mL being the most effective and doses under 0.1 mg/mL having minimal effect. IVIG diminished the number of stimulated cells progressing in the cell cycle by 30%, and there was no difference in cell viability between IVIG-treated and IVIG-untreated cells. The production of IgE in culture by anti-CD40/IL4-stimulated B lymphocytes was curtailed by greater than 80% after addition of 5 mg/mL IVIG. This was associated with a decrease in IgE (epsilon) transcripts in IVIG-treated cultures. CONCLUSION These data indicate that diminution of IgE production in anti-CD40/IL-4-stimulated B cells by IVIG is due to inhibition of early events related to proliferation and progression in the cell cycle.

Dose-dependent agonist and antagonist effects of the platelet-activating factor analogue 1-palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine on B lymphocytes.

BACKGROUND Platelet activating-factor (PAF), an ether-linked phospholipid, is a potent activator of B lymphocyte cell lines. The related ester-linked phospholipid, 1-palmitoyl-2-acetoyl-sn-glycero-3-phosphocholine (PAGPC), is synthesized by tissues important in B-cell development. OBJECTIVES We examined whether PAGPC was capable of influencing immunoglobulin synthesis in B lymphocytes and compared its action with that of PAF. We also examined the interaction of the two mediators as agonists or competitive antagonists. METHODS Ramos, an IgM-secreting immature B-cell line that expresses PAF receptor, was used in these experiments. The effect of PAF, PAGPC, or both mediators together on IgM secretion and anti-IgM-mediated apoptosis was measured. RESULTS Both PAF and PAGPC stimulated IgM production in Ramos cells in a dose-dependent fashion, with PAGPC being approximately three logs less potent than PAF. The effect of both mediators was inhibited by specific PAF receptor antagonists. Preincubation with suboptimal concentrations of PAGPC inhibited the ability of PAF to increase IgM secretion by Ramos cells. Additionally, preincubation with low concentrations of PAGPC prevented PAF from rescuing Ramos cells from apoptosis induced by cross-linking the B-cell receptor with anti-IgM antibodies. PAGPC caused PAF receptor desensitization because displacement of bound PAGPC with high concentrations of bovine serum albumin did not reverse its PAF antagonist effect. CONCLUSIONS PAF and PAGPC are biologically active phospholipids, but PAF is approximately 1000 times more potent. At high concentrations, PAGPC acts similarly to PAF, whereas at lower concentrations, PAGPC acts as a functional PAF antagonist. Because it is secreted at sites of inflammation and allergic reactions, PAGPC may be an endogenous regulator of the effects of PAF.

Platelet-Activating Factor Antagonists Decrease Follicular Dendritic-Cell Stimulation of Human B Lymphocytes

Both B-lymphoblastoid cell lines and tonsillar B lymphocytes express receptors for platelet-activating factor (PAF). In lymph node germinal centres, B lymphocytes interact with follicular dendritic cells (FDCs), which present antigen-containing immune complexes to B lymphocytes. FDCs have phenotypic features that are similar to those of stromal cells and monocytes and may therefore be a source of lipid mediators. In this study, we evaluated the effects of the PAF antagonist WEB 2170 on the activation of tonsillar B lymphocytes by FDCs. FDCs were isolated from tonsils by Bovine Serum Albumin (BSA) gradient centrifugation. After being cultured for 6 to 10 days, they were incubated with freshly isolated B cells in the presence or absence of the specific PAF receptor antagonist WEB 2170. B-lymphocyte proliferation was assessed by [3H]-thymidine incorporation, and immunoglobulin (Ig) G and IgM secretion was assessed by enzyme-linked immunosorbent assay (ELISA). WEB 2170 (10-6 to 10-8 M) inhibited [3H]-thymidine incorporation by up to 35% ± 3%. Moreover, the secretion of IgG and IgM was inhibited by up to 50% by WEB 2170 concentrations ranging from 10-6 to 10-8 M. There was no evidence of toxicity by trypan blue staining, and the addition of WEB 2170 to B cells in the absence of FDCs did not inhibit the spontaneous production of IgG or IgM. The effect of the PAF antagonist is primarily on B lymphocytes, as reverse transcription polymerase chain reaction detected little PAF receptor messenger ribonucleic acid (mRNA) from FDCs. These data suggest that endogenous production of PAF may be important in the interaction of B lymphocytes with FDCs.