Quantitative, rather than qualitative, differences in CD69 upregulation in human blood eosinophils upon activation with selected stimuli

Background: The study aimed to investigate whether CD69 expression on granulocytes is subject to specific regulation by inflammatory mediators, and, if so, to identify these factors in relation to eosinophil activity markers such as the EG2 epitope and ECP release. Methods: Peripheral blood leukocytes from healthy donors were used. The surface and intracellular distribution of CD69 was investigated with a whole-blood cell-membrane permeabilization technique, the FOG method, and flow cytometry. In vitro stimulation was performed with GM-CSF, IL-5, IL-5 plus eotaxin, LPS, and fMLP. Results: A preformed intracellular pool of CD69 was demonstrated in both eosinophils and neutrophils, but not in monocytes. Almost no resting eosinophils, neutrophils, or monocytes expressed CD69 on the cell surface. However, in vitro stimulation with selected stimuli increased the proportion of CD69-positive eosinophils to various extents: with GM-CSF being the most and fMLP the least efficient stimulus. The neutrophils did not respond under these conditions. Increased expression of the EG2 epitope and initiation of degranulation preceded CD69 upregulation. Conclusions: Eosinophils and neutrophils from healthy donors have a preformed intracellular pool of CD69, which is mobilized on the cell surface on eosinophils, but not on neutrophils, to various extents by selected stimuli. Monocytes, however, do not have a preformed intracellular pool of CD69. Our data indicate that a kinetic order exists among the EG2 expression, the degranulation process, and CD69 upregulation. Due to a quantitative, rather then a qualitative, upregulation of CD69 by stimuli associated with both allergic and bacterial inflammation. CD69 may be a potential activity marker of clinical value.