Mechanisms of Eosinophil Cationic Protein Release in the Serum: Role of Adhesion Molecules

Abstract

Background: Measurement of eosinophil cationic protein (ECP) in serum has been utilized as a marker for allergic inflammation. The serum level of ECP represents the level found in vivo plus additional proteins released in vitro from peripheral blood eosinophils during the coagulation period. The mechanisms of release, however, are unclear. We investigated a possible involvement of adhesion molecules in the ECP release. Materials and Methods: Venous blood was drawn in the presence of EDTA from allergic donors. The blood was incubated with neutralizing monoclonal antibodies to CD18, CD11a, CD11b, CD29, CD49d, CD54, α₄β₇, or isotype-matched control antibodies, respectively, at 4°C for 30 min. Calcium gluconate (calcium) was then added to induce coagulation. The blood was further incubated for 90 min and centrifuged to obtain the serum. ECP in the serum was measured with RIA. In some experiments, purified eosinophils were incubated with plasma and calcium, then ECP in the supernatants was assayed. Results: ECP in the samples with calcium was significantly higher than in those without calcium. Purified eosinophils released ECP upon plasma coagulation. Anti-CD18, CD49d, and α₄β₇ antibodies significantly suppressed ECP levels in the serum. Conclusions: These results suggest that ECP release in the serum is calcium and plasma coagulation-dependent and that cell adhesion through α_Lβ₂, α_Mβ₂, α₄β₁ and α₄β₇ integrins is at least in part responsible for ECP release.