Production of Human Antibodies to Bee Venom Phospholipase A₂ in Vitro

Abstract

Phospholipase A2 (PLA) is the major antigen of bee venom. Whereas individuals frequently stung by bees, such as bee keepers, show high levels of IgG4 anti-PLA antibodies in serum, most patients sensitive to bee venom possess increased IgE anti-PLA. We have established a culture system by which anti-PLA antibodies can be induced in vitro. Peripheral blood mononuclear cells were stimulated in a first step with PLA and/or pokeweed mitogen (PWM). After 3 days of culture the cells were washed and further incubated with fresh medium. Anti-PLA antibodies were estimated by an enzyme-linked immunosorbent assay (ELISA) and anti-PLA antibody secreting cells were counted by means of an ELISA plaque assay. Cells from bee keepers, but not those from normal donors, produced anti-PLA IgG in vitro. The isotype pattern of anti-PLA antibodies produced in vitro was identical to that found in serum of the respective donor. Anti-PLA IgM antibody-secreting cells developed at low frequencies in both bee keepers and normal donors whereas PLA-specific IgE could not be detected. The formation of anti-PLA IgG was suppressed by high amounts of antigen, while the IgM response was not affected. We conclude that PWM-induced antibodies reflect the in vivo situation of the donors and that anti-PLA IgG and IgM are regulated by different pathways and mechanisms.

Bee venom phospholipase A2‐specific T cell clones from human allergic and non‐allergic individuals: cytokine patterns change in response to the antigen concentration

“…Hyposensitization of individuals allergic to bee sting is a standard clinical procedure 149, 501. The PLA-specific IgE/IgG4 ratio in serum decreases with prolonged administration of bee venom to allergic patients [38,40,[51][52][53]. Since antibody production is regulated by Tcell-derived factors, it is rcasonable to speculate that elevated antigen levcls, artificially maintained in allergic patients during hyposensitization, can modify the cytokine pattcrn of specific T cells and as a Consequence the TgE/lgG4 ratio.…”

Epitope-specific T cell tolerance to phospholipase A2 in bee venom immunotherapy and recovery by IL-2 and IL-15 in vitro.

“…The IgE and IgG4 anti-PLA antibody contents in patients serum and culture supernatants were measured in duplicates by ELISA (34)(35)(36). PLA-specific antibodies were detected in 0.5 g/well PLA-coated ELISA plates (Maxisorb) which were incubated with patients' serum and culture supernatants at different dilutions.…”

Regulation of the Cytokine Production of Allergen-Specific Human T-Cell Clones by the Allergen

“…PLA (Sigma-P9279) was reduced and carboxymethylated to re move enzymatic activity and associated cell-toxic effects as previously described [10], The establishment and maintenance of PLA-specific T-cell clones and ELISA for IL-4 and IFN-y are described in detail elsewhere [11]. Triplicate samplesof 3 x 10JPLA-specificT-cell clones were incubated in 2(X) ul of 96-well microtiter plates for 48 h at 37°C, 5% CO; with equal amounts of autologous 6,000-rad-irradiated Epstein-Barr virus transformed B (EBV-B) cells in the presence of different amounts of PLA.…”