Effect of levamisole on alkaline phosphatase activity and immunoglobulin secretion of lipopolysaccharide-stimulated murine splenic lymphocytes

Abstract

Background: Alkaline phosphatases (APase) are a group of enzymes whose activity increases above normal levels in conditions of diseases such as cancer. In the present study, the role of APase using mitogen-stimulated murine lymphocytes was investigated. Methods: U266B1 cells and RPMI 8226 cultures were kept at 37°C in a humidified incubator with 5% CO2. The cultures were pulsed with 0.5 μCi of 3H-thymidine and were harvested onto glass fiber filter using Skatron automatic cell harvester. The dried filters were transferred into toluene-based scintillation cocktail, and the radioactivity was measured using Beckman scintillation counter. APase activity was determined by p-nitrophenol phosphate hydrolysis. The intracellular immunoglobulin E (IgE) content was quantified by western blot assay. U266 B1 and RPMI 8226 were cultured at 0.25 × 106/ml with and without 1 mM levamisole for 48 h, and 15 ml of the culture supernatant was collected and lyophilized. Electrophoresis was carried out on 30 μl of the dialyzed samples. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was performed and the gels were silver stained. Results: APase may be involved in the constitutive proliferation as well as in Ig secretion of myeloma cells. It was observed that murine splenic lymphocytes showed an increase in proliferative response concomitant with an increase in the APase activity and Ig secretion upon mitogenic stimulation (0.5–2.5 mM, P > 0.05). Conclusion: Levamisole significantly inhibited the APase activity when added to the lipopolysaccharide-stimulated cells at the initiation of the culture. Significance of the present study is discussed in the light of existing literature.

Keywords

• Immunoglobulin secretion
• levamisole
• mitogenic stimulation
• myeloma cells