Weak bases increase surface IgM expression in 70Z/3 B lymphoid cell line without increasing kappa gene expression.
A murine B lymphoid cell line, 70Z/3, has proven to be a useful model to study the developmental transition from membrane immunoglobulin (mIgM)-negative to mIgM-positive B cells. 70Z/3 cells have normal intracellular levels of mu heavy chain but kappa mRNA is transcribed at a very low rate. Because both kappa and mu chains are required for IgM to localize on the external membrane the cells remain essentially mIgM negative. A number of biologic agents can stimulate kappa mRNA transcription leading to increased surface IgM expression. Pharmacologic agents which cause cytoplasmic alkalinization have also been shown to increase the level of surface IgM expression when assessed by FITC-labeled anti-IgM antibodies. This increase has been used to argue that cytoplasmic alkalinization is sufficient to stimulate 70Z/3 differentiation but previous studies did not measure kappa mRNA levels or rate of synthesis. In this study we set out to determine whether the increased FITC staining that follows cytoplasmic alkalinization results from increased surface IgM expression and whether that increase results from activation of kappa mRNA transcription. When cells were treated with methylamine, NH4Cl, or monensin there was transient cytoplasmic alkalinization. The levels of surface IgM expression were measured by flow cytometry of cells stained with FITC-labeled anti-mouse kappa and mu. Using this criterion, a 24-hr treatment with either weak bases or monensin increased the level of surface IgM. The increase in fluorescence was not the result of nonspecific binding or uptake by the cell because there was no increase in fluorescence when methylamine-treated cells were stained with FITC-labeled antibody directed against an antigen not found on these cells. Iodination of surface proteins confirmed that the increase in fluorescence was the result of increased levels of IgM protein on the cell surface. However, exposure of the cells to weak bases or monensin caused no increase in either the steady-state level of kappa light chain mRNA, or in the level of kappa protein. We conclude that the transient alkalinization is not sufficient to induce differentiation of the 70Z/3 cells.
Ostrowski
J
J
Department of Medicine, University of Washington, Seattle 98195.
Sibley
C H
CH
Stepinski
J
J
Stanton
T H
TH
Smith
L L
LL
Bomsztyk
K
K
D000643:Ammonium Chloride / Q000494:pharmacology*; D000818:Animals; D001402:B-Lymphocytes / Q000378:metabolism*; D015153:Blotting, Western; D002451:Cell Compartmentation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects*; D005803:Genes, Immunoglobulin; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007075:Immunoglobulin M / Q000378:metabolism*; D007145:Immunoglobulin kappa-Chains / Q000235:genetics* / Q000378:metabolism; D066298:In Vitro Techniques; D008744:Methylamines / Q000494:pharmacology*; D051379:Mice; D008985:Monensin / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D011947:Receptors, Antigen, B-Cell / Q000378:metabolism*; D012964:Sodium / Q000502:physiology
D007075:Immunoglobulin M; D007145:Immunoglobulin kappa-Chains; D008744:Methylamines; D012333:RNA, Messenger; D011947:Receptors, Antigen, B-Cell; D000643:Ammonium Chloride; D008985:Monensin; D012964:Sodium; C027451:methylamine