Regulation of human basophil function by phosphatase inhibitors

Abstract

Okadaic acid, a cell permeant inhibitor of protein serine/threonine phosphatases (PPs), attenuated the IgE‐mediated release of the pre‐formed mediator, histamine from human basophils in a time‐ and dose‐dependent manner. Optimal inhibition (77 ± 4%, P < 0.0001) of histamine release was observed following a 2 h incubation with 1 μm okadaic acid. Okadaic acid and two analogues of okadaic acid were also studied and were found to inhibit the IgE‐dependent release of histamine. Concentrations required to inhibit release by 50% (IC50) were 0.6 μm for okadaic acid and 7.5 μm for okadaol, whereas okadaone was inactive. The structurally‐unrelated PP inhibitor, calyculin A, also inhibited IgE‐dependent histamine release from basophils dose‐dependently and was approximately six fold more potent than okadaic acid. The IgE‐mediated generation of sulphopeptidoleukotrienes (sLT) from basophils was inhibited by okadaic acid and related analogues with the following rank order of potency; okadaic acid (approx. IC50 0.3 μm) > okadaol (3 μm) > okadaone (inactive). Okadaic acid, okadaol and okadaone (all at 3 μm) inhibited the IgE‐mediated generation of the cytokine interleukin 4 (IL4) from human basophils by 67 ± 9% (P < 0.002), 48 ± 14% (P < 0.05) and 8 ± 7% (P = 0.31), respectively. Extracts of purified human basophils liberated 32P from radiolabeled glycogen phosphorylase and this PP activity was inhibited by 17 ± 3% (P < 0.0005) by a low (2 nM) concentration of okadaic acid and was inhibited by 96 ± 1% (P < 0.0001) by a higher (5 μm) concentration of okadaic acid. Because a low (2 nM) concentration of okadaic acid inhibits PP2A selectively whereas a higher (5 μm) concentration inhibits both PP1 and PP2A, these findings suggest that both PP1 and PP2A are present in basophils. In total these data suggest that PPs are resident in human basophils and that PPs may be important in the regulation of basophil function.

Preliminary characterization of the role of protein serine/ threonine phosphatases in the regulation of human lung mast cell function

“…Our own previous studies have shown that okadaic acid inhibits the stimulated release of mediators from human lung mast cells (HLMC) suggesting that PPs may regulate HLMC responses (Peachell & Munday, 1993). Similar studies in rat peritoneal mast cells (Takei et al, 1993;Estevez et al, 1994), rat basophilic leukaemia cells (Sakamoto et al, 1994) and human basophils (Botana & MacGlashan, 1993;Peirce et al, 1996) have also been reported. In the present work we have attempted to characterize PP activities in HLMC and to determine whether okadaic acid inhibits HLMC responses by interacting with PPs.…”

Characterization of protein serine/threonine phosphatase activities in human lung mast cells and basophils

“…An attractive feature of okadaic acid is that it is cell‐permeant and this property has been exploited advantageously to establish whether PPs might be involved in regulating cellular processes (Haystead et al , 1989; Hardie et al , 1991; Hardie, 1993). We, ourselves, have studied okadaic acid, and have shown it to be an effective inhibitor of IgE‐mediated histamine release from HLMC (Peachell & Munday, 1993; Peirce et al , 1997) and basophils (Peirce et al , 1996).…”

“…We and others have previously reported that okadaic acid is an effective inhibitor of mediator release from human lung mast cells (HLMC) and basophils (Botana & MacGlashan, 1993; Peachell & Munday, 1993; Peirce et al , 1996, 1997). Moreover, we have reported that extracts of HLMC and basophils contain PP activities which are sensitive to okadaic acid.…”