Eosinophils Maintain Their Capacity to Signal and Release Eosinophil Cationic Protein Upon Repetitive Stimulation with the Same Agonist

ECP is released after priming with GM-CSF and subsequent stimulation with PAF or C5a, but not eotaxin. Data presented as mean ± SEM. The numbers of independent experiments for each condition varied between two and six (∗, p < 0.05).

Release of ECP from eosinophils upon repeated stimulation. GM-CSF-primed eosinophils were stimulated with either PAF or with C5a twice. Time periods between stimulations included a washing step and varied from 0 to 30 min (PAF) and from 0 to 80 min (C5a). No significant ECP releases upon second stimulation were observed within these time periods without a washing step after the first stimulation (ECP concentrations always <15 μg/L). Results of one representative of five independent experiments is shown in each case.

Intracellular calcium changes in eosinophils in response to repeated stimulation with PAF or with C5a. Calcium signals were measured by fura-2 fluorescence. A, Arrows indicate the time point of addition of the agonists. The cells were not washed between the stimulations. One representative of five independent experiments is shown in each case. B, Calcium signals upon repeated stimulation. The cells were washed between the applications of the agonists. “0” represents freshly purified cells that had been stimulated with the indicated agonists for the first time. The time interval (5 and 15 min) between the applications is indicated and includes the washing step. Five minutes after the first stimulation, a second PAF stimulation was associated with rapid calcium response, which was only slightly less compared with the first response. In contrast, complete homologous desensitization was observed in the case of C5a, because the second C5a stimulation did not result in an increase of intracellular free calcium levels at this time point. Results of one representative of five (C5a) or six (PAF) independent experiments is shown.

Expression of PAF receptors on eosinophils. A, Flow cytometry. Cells were incubated without (upper left) or with (upper middle and right) the fluorescent PAF agonist, at 4° or 37°C as indicated. The cells were then washed and analyzed again by FACS (lower left and middle). The results indicate that the washing procedure fully removed the PAF agonist from cells incubated at 4°C but not at 37°C. Reapplication of the fluorescent probe (lower right) indicates the availability of receptors following the washing procedure. The number at the upper right corner of each histogram indicates the ratio calculated as the mean channel fluorescence level following PAF staining divided by the mean channel fluorescence level obtained from unstained eosinophils. In the presence of WEB 2086 (1 mM), fluorescent PAF binding was blocked (ratio: 0.96). Results are representative of three independent experiments. B, Fluorescent microscopy. Fluorescent lyso-PAF bound to eosinophil membrane. Washing removed the fluorescent ring signal. A second incubation with fluorescent lyso-PAF resulted again in a ring-like staining of the eosinophils, suggesting the availability of free PAF receptors. Similar results were obtained using fluorescent PAF, although the fluorescent signal was slightly decreased compared with lyso-PAF. Results are representative of three independent experiments.

The effect of GM-CSF on ECP mRNA in control eosinophils (A) or following partial depletion of ECP levels due to concomitant stimulation with both PAF and C5a (B). G3PDH served as control. Results are representative of three independent experiments.

The effect of GM-CSF on ECP protein expression in eosinophils with decreased ECP levels. Eosinophils were simultaneously three times stimulated with optimal concentrations of PAF and C5a within 6 h. The following percentages of the original ECP content were observed in the five presented independent experiments: 1, 76%; 2, 52%; 3, 30%; 4, 53%; and 5, 40%. The viability of the eosinophil populations after the releases was 80–95%. Eosinophils were washed and cultured in the presence and absence of GM-CSF for 14 h and total ECP contents determined.