Hydrolysis of inositol phospholipids induced by stimulation of the T cell antigen receptor complex in antigen-specific, murine helper T cell clones. Requirement for exogenous calcium.
Two murine, keyhole limpet hemocyanin-specific, Th cell clones were studied for their ability to respond to antibody-mediated stimulation of the TCR complex or to Ag-pulsed accessory cells by hydrolyzing inositol phospholipids. Both clones were positive for the determinant expressed on the epsilon chain of CD3 that is recognized by the mAb, 145-2C11 (2C11 mAb); one clone also expressed the V beta 8 epitope of the alpha/beta chains of the TCR recognized by the F23.1 mAb. Treatment of these cells with 2C11 or F23.1 mAb adsorbed onto polystyrene beads induced a time-dependent accumulation of inositol phosphates (IP). Keyhole limpet hemocyanin-pulsed accessory cells which expressed the appropriate MHC phenotype also induced IP accumulation, whereas no response was induced by medium-treated or MHC congenic accessory cells. The hydrolysis of inositol phospholipids induced by TCR perturbation depended upon the presence of exogenous Ca2+; Mg2+ did not substitute for Ca2+. Treatment of cells with ionomycin at concentrations up to 30 microM was unable to induce hydrolysis of inositol phospholipids, indicating that entrance of Ca2+ was itself insufficient to generate IP. Stimulated IP generation was rapidly blocked upon addition of EGTA to the incubation medium. Reducing the level of exogenous Ca2+ decreased the production of inositol mono-, bis-, and trisphosphate isomers similarly, suggesting that extracellular Ca2+ was required for the initiation of the hydrolysis rather than affecting phospholipase C affinity for its substrates. We concluded that activation of inositol phospholipid hydrolysis by perturbation of the TCR complex in the Th cell clones under investigation displays a Ca2+-dependent component which is likely to be proximal to IP generation.
Bonvini
E
E
Laboratory of Cell Biology, US Food and Drug Administration, Bethesda, MD 20892.
DeBell
K E
KE
Kolber
M A
MA
Hoffman
T
T
Hodes
R J
RJ
Taplits
M S
MS
D000818:Animals; D000911:Antibodies, Monoclonal / Q000502:physiology; D000938:Antigen-Presenting Cells / Q000276:immunology / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D002999:Clone Cells / Q000145:classification / Q000187:drug effects / Q000378:metabolism; D000939:Epitopes / Q000276:immunology*; D006433:Hemocyanins / Q000276:immunology; D006868:Hydrolysis; D051379:Mice; D008810:Mice, Inbred C57BL; D008863:Microspheres; D010716:Phosphatidylinositols / Q000276:immunology / Q000378:metabolism*; D011948:Receptors, Antigen, T-Cell / Q000187:drug effects / Q000378:metabolism*; D006377:T-Lymphocytes, Helper-Inducer / Q000145:classification / Q000187:drug effects / Q000378:metabolism*
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010716:Phosphatidylinositols; D011948:Receptors, Antigen, T-Cell; D006433:Hemocyanins; C032808:keyhole-limpet hemocyanin; D002118:Calcium