Pharmacological investigations with different protein kinase C inhibitors on IgE-dependent and IgE-independent activation of human basophils

Abstract

In the present study different selective inhibitors of the multifunctional serine/threonine kinase protein kinase C (PKC) were investigated on classical activation pathways of basophils in comparison to the nonselective protein kinase inhibitor staurosporine. The potent inhibitors Ro 31-7549, Ro 31-8220, calphostin C and ilmofosine (BM 41.440), which show selectivity for PKC in vitro, significantly potentiated Fc epsilon RI-mediated histamine release up to 50% vs. controls at concentrations > 10(-7) mumol/l but were without any intrinsic histamine releasing activity. Direct activation of cellular PKC by phorbol ester was suppressed by all compounds apart from ilmofosine at the same concentrations. We did not observe statistically significant effects of selective PKC inhibitors on exocytosis induced by the peptide formylmeth-leu-phe (FMLP) or the ionophore A23187, whereas staurosporine potentiated the FMLP-induced histamine release in a dose-dependent fashion: maximum potentiation was 63.5 +/- 8.9% vs. control at 1 mumol/l (n = 4). The findings suggest that PKC exhibits differential functions during biochemical events of stimulus-secretion coupling in human basophils supposedly by its distinct subtypes. With respect to the present data, TPA-induced and IgE-mediated signals are not closely correlated.

Activation and inhibition of mediator release from skin mast cells: a review of in vitro experiments

“…The PKC inhibitors Ro 31-7549, Ro 31-8220, and calphostin C induced a dose-dependent inhibition of IgE-mediated histamine release from isolated human skin mast cells (manuscript in preparation). These results are in contrast to those from investigations with basophils showing a dosedependent non-cytotoxic potentiation of IgE-mediated histamine release reflecting the biochemical heterogeneity between both cell types [70,71]. Further evidence for a differential role of PKC (isozymes) comes from experiments with the phorbol ester TPA, a strong ligand and activator ofPKC.…”

Heterogeneity of Human Skin Mast Cells and Human Basophils

“…Peripheral basophils were isolated after venipuncture from healthy donors by dextran sedimentation as previously described [9,17], In some experiments buffy coats resulting from dextran sedimentation (4 ml) were layered on top of 3 ml Ficoll-Paque (Pharmacia, Uppsala, Sweden) in 12-ml conical plastic tubes (Sarstedt) and centrifuged at 300 g for 25 min (room temperature). The lymphocyte/monocyte/basophil fraction at the interface was diluted with buffer A to 10 ml and was incubated for 10 min (room temperature, turntable) with 200 pi of CD2+, CD9+ and CD 19+ immunomagnetic beads (Dynal, Hamburg, Germany) to reduce T cells, mono cytes, macrophages, and B cells by a magnetic particle concentrator (Dynal).…”

“…The cellular and the supernatant fractions were stored at -2 0°C until determination. Histamine release was de termined in both supernatants and cell pellets using a fluorometric autoanalyzer [9,17]. Histamine release was expressed as a percentage of the total histamine initially in the cells, corrected for the spontaneous release occurring in the absence of secretagogue.…”