Detection of functional platelet-activating factor receptors on human tonsillar B lymphocytes.

Although platelet-activating factor (PAF) receptors have been found on B lymphoblastoid cell lines, the action of PAF on freshly isolated human B cells has not been clearly demonstrated. Using a sensitive semiquantitative reverse-transcriptase PCR, we have found PAF receptor mRNA expressed by tonsillar B lymphocytes, but little in T lymphocytes. Examination of Percoll-fractionated tonsillar B cells indicated that the low density (primarily germinal center cells) and medium density fractions had approximately twofold more PAF receptor mRNA relative to the high density fraction. PAF (10-7 M) stimulated increases in intracellular Ca2+ that were consistently higher in the low and medium density B lymphocytes compared with high density cells. The PAF receptor antagonist Web 2170 inhibited this. Addition of PAF, but not lyso- or enantio-PAF, induced four- to sixfold greater synthesis of IgM and IgG in low and medium density cells compared with unstimulated controls, but had little effect on Ig production by high density cells. To investigate how PAF may influence Ig synthesis, PAF-stimulated B cells were examined for production of the Th2-type cytokines IL-4 and IL-13. PAF induced IL-4 and IL-13 mRNA expression in 17% of CD20+ cells, and IL-4 was detected in cell supernatants after 48-72 h of culture. Together, these data strongly suggest that functional PAF receptors are expressed on B cells in tonsils.

Characterization of B lymphocytes rescued from apoptosis by platelet-activating factor.

B lymphocyte development is characterized by deletion via apoptosis of immature cells that are insufficiently stimulated. We have previously demonstrated that crosslinking of the B cell receptor (BCR) using anti-IgM antibodies (alphaIgM) (2 microg/ml) in Ramos B lymphoblastoid cells causes deletion of 30-40% of cells by apoptosis in 24 h. Addition of the potent lipid mediator platelet-activating factor (10(-7) M) to alphaIgM stimulated Ramos cells significantly decreases the number of apoptotic cells as measured by annexin V labeling. We have characterized the phenotype of Ramos cells that have not become apoptotic following BCR stimulation. In these cells, there is a significant decrease in the surface expression of the VLA-4 adhesion molecule (31% of control expression) and surface IgM expression (sIgM) (53% of control expression). Significantly fewer cells co-incubated with platelet-activating factor (PAF) underwent apoptosis, and the remaining cells maintained control levels of VLA-4 (104% of control expression) and sIgM expression (104% of control). All of these protective effects were inhibited by the specific PAF receptor antagonist, WEB 2170. The action of PAF on alphaIgM induced apoptosis was not inhibited by either cycloheximide or cytochalasin B, suggesting that de novo protein synthesis and F-actin polymerization were not implicated in the rescue of Ramos cells by PAF. In contrast, the ability of PAF to maintain sIgM and VLA-4 expression at control levels was inhibited by cycloheximide (7. 5 microg/ml). Cytochalasin B (5 microg/ml) had no effect on sIgM expression but blocked the decrease in VLA-4 expression mediated by alphaIgM. These data indicate that PAF's effect on rescuing and maintaining alphaIgM stimulated Ramos B cells is mediated via at least two pathways. Abrogation of apoptosis does not require de novo protein synthesis, while maintenance of sIgM and VLA-4 expression requires protein synthesis.

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.

Platelet-activating factor abrogates apoptosis induced by cross-linking of the surface IgM receptor in a human B lymphoblastoid cell line.

B lymphocyte development is characterized by deletion, via apoptosis, of immature cells that are stimulated via the B cell receptor in the absence of a second signal. We have investigated whether platelet-activating factor (PAF), a potent B lymphocyte activator, can provide a complementary signal with B cell receptor ligation to abrogate apoptosis. Cross-linking of the surface IgM on Ramos B lymphoblastoid cells using anti-IgM Abs (2 microg/ml) caused programmed cell death in 34 +/- 5.4% of the cells. Coincubation of PAF (10(-7)M) with alphaIgM led to a significant decrease in apoptotic cells as measured by DNA laddering and TUNEL assay (13.8 +/- 3%). The effect of PAF was dose dependent (10(-7)-10(-9) M) and was inhibited by the specific PAF receptor antagonist, WEB 2170. PAF protected cells from the effect of alphaIgM for up to 1 h after it was added. alphaIgM-induced programmed cell death in Ramos cells was blocked by catalase and, therefore, is caused in part by the production of toxic hydroxyl radicals from hydrogen peroxide. We investigated the action of PAF on markers of intracellular oxidation. H2O2 in low doses induced apoptosis, via production of OH. radicals. PAF inhibited H2O2-induced apoptosis in Ramos cells; it also attenuated H2O2- and alphaIgM-mediated increases in hydroxyl radical (OH.) as measured by the oxidation of 2',7'-dichlorofluorescein diacetate to 2',7'-dichlorofluorescein and blocked the depletion of reduced glutathione induced by alphaIgM. PAF maintained IgM secretion, which was greatly inhibited by incubation with alphaIgM alone. These data indicate that PAF potentially provides an important cosignal to surface IgM-stimulated Ramos cells by inhibiting apoptosis. This is in part due to the activity of PAF in the oxidant/ antioxidant pathway.