Cytokine RANTES released by thrombin-stimulated platelets is a potent attractant for human eosinophils

Abstract

Thrombin stimulation of human platelets results in the release of a preformed proteinaceous human eosinophil (Eo)-chemotactic activity. By the use of different high-performance liquid chromatography techniques, two Eo-chemotactic polypeptides (EoCPs), tentatively termed EoCP-1 and EoCP-2, were purified to homogeneity. Upon SDS-PAGE analysis, these chemotaxins showed molecular masses near 8 kD. NH2-terminal amino acid sequence analysis revealed identical sequences for both EoCP-1 and EoCP-2, which are also identical to that of RANTES, a cytokine that structurally belongs to the interleukin 8 superfamily of leukocyte selective attractants, and that is known to be a "memory-type" T lymphocyte-selective attractant. In the major Eo chemotaxin, EoCP-1, the residues 4 and 5, which in EoCP-2 were found to be serine residues, could not be identified. Electrospray mass spectrometry (ESP-MS) of EoCPs revealed for EoCP-2 a molecular mass of 7,862.8 ± 1.1 daltons, which is 15.8 mass units higher than the calculated value of RANTES, indicating that EoCP-2 is identical to the full-length cytokine, and oxygenation, probably at methionine residue number 64, has taken place. Upon ESP-MS, EoCP-1 showed an average molecular mass of 8,355 ± 10 daltons, suggesting O-glycosylation at these serine residues. Both natural forms of RANTES showed strong Eo-chemotactic activity (ED50 = 2 nM) with optimal chemotactic migration at concentrations near 10 nM, however, there were no significant migratory responses with human neutrophils. Chemotactic activity of RANTES for human Eos could be confirmed using recombinant material, which has been found to be as active as the natural forms. Since RANTES gene expression has been detected in activated T lymphocytes, and recombinant RANTES was shown to be a "memory" T lymphocyte-selective attractant, it is now tempting to speculate about an important role of RANTES in clinical situations such as allergene-induced late-phase skin reactions in atopic subjects or asthma, where in affected tissues both memory T cells and Eos are characteristic.