Mechanisms of human eosinophil activation by complement protein C5a and platelet-activating factor: similar functional responses are accompanied by different morphologic alterations.

Abstract

The complement system is an important amplification system for the propagation of allergic as well as pseudoallergic inflammatory reactions. In the present study, the effect of the major anaphylatoxin C5a was compared with that of platelet-activating factor (PAF) on highly purified eosinophils (> or = 95%) by functional as well as morphologic criteria. Upon stimulation with C5a, eosinophils maintained their spheric structure, developing short, pseudopodia-like protrusions, whereas PAF induced the generation of a number of digitating protrusions. As shown by functional and ultrastructural assay systems, both stimuli provoked significant extracellular and intracellular H2O2 production in eosinophils, which was inhibited by cytochalasin B. With C5a, a pronounced H2O2 production was detected within the small cytoplasmic vesicles, whereas PAF-induced H2O2 production was observed on the outer surface of the plasma membrane in the contact zones between adjacent cells. Morphologic signs of degranulation induced by C5a and PAF were accompanied by the significantly increased release of eosinophil cationic protein and eosinophil peroxidase in the presence of cytochalasin B. Like PAF, C5a induced a significant production of reactive oxygen species in eosinophils, as measured by lucigenin-dependent chemiluminescence (CL) responses in eosinophils. Maximal responses, comparable with those of interleukin-5 (100 U/ml), were observed at concentrations of 10(-5)-10(-6) and 10(-7)-10(-8) M for PAF and C5a, respectively. Separation of eosinophils by discontinuous density gradients revealed the existence of two hypodense eosinophil populations, one of them showing significantly reduced CL responses upon stimulation with C5a and PAF. In addition, CL responses upon stimulation with C5a and PAF were abrogated by cytochalasin B, staurosporine, and wortmannin, and were almost completely blocked by pertussis toxin. In conclusion, these data indicate that C5a induces events in human eosinophils comparable to those induced by PAF in the assay systems tested. Thus, C5a, generated after activation of the complement system, may be of major importance for the eosinophil activation observed in eosinophil-related disease.