Characterisation of eosinophil activity markers : relation to allergic inflammation and apoptosis

An allergic inflammation is characterised by many different factors, among which eosinophils and the release of their granule content e.g. eosinophilic cationic protein (ECP) constitute main features. Activated eosinophils play an important role in both the early and late stages of the allergic inflammation and participate in the remodelling process in tissue, thus contributing to scarring and fibrosis development. Moreover, there are data showing that eosinophils in an activated state have prolonged survival, which could in part be explained by a decrease in apoptosis.

This thesis focuses on identification of eosinophil activity markers of importance for the clinical evaluation of allergic inflammation, as well as the relation between activation and prolonged survival of the eosinophil granulocyte.

We have shown that when analysing activated cells, primarily eosinophils, the choice of anticoagulant is of importance for the maintenance of cell survival and function, and that citrate as anticoagulant is preferable to EDTA. These studies were designed to allow detection of eosinophil activity markers, both in vitro (the intracellular expression of the EG2-epitope on ECP (EG2), ECP release as well as CD69 expression) and in vivo (eosinophil number, ECP, EG2 and CD69). When examining the kinetic relation between EG2 expression, ECP release, CD69 upregulation and apoptosis, we found that EG2 expression and ECP release precede CD69 upregulation and apoptosis, but we were not able to establish any relation between CD69 upregulation and level apoptosis susceptibility. However, one must take in account that the presence of the natural ligand for CD69, which was still unknown at the time of the investigation, may play a role in induction of apoptosis.

To further examine the apoptosis process in eosinophils, we analysed eosinophils from allergic and non-allergic individuals, and we found an increased survival in cells from allergic individuals as compared to non-allergic individuals. In addition, we examined the possible role of the mitochondrial membrane potential, release of cytochrome c and caspase 3 activation. When we used the apoptosis inducer tributyltin (TBT), an extremely fast induction of apoptosis was seen (measured by caspase 3 activity and Annexin binding) without the prior mitochondrial changes generally associated with apoptosis (dissipation of the mitochondrial membrane potential and release of cytochrome c).

In conclusion, we have identified different markers, which can be used for detection of activated eosinophils in relation to the allergic inflammation. We have also described the kinetic relation between markers for activation, degranulation and apoptosis m eosinophils. Our hope is that these results may eventually contribute to the identification of a drug that can selectively induce apoptosis in eosinophils without causing activation and degranulation, thus decreasing the pathophysiological effect of eosinophils both in the acute inflammation, as well as in the process of tissue remodelling after injury.

List of scientific papers

I. Hallden G, Nopp A, Ihre E, Peterson C, Lundahl J (1999). Conditions in blood sampling procedures that extend the ex vivo stability of eosinophil activity markers in peripheral blood from allergic patients and healthy controls. Ann Allergy Asthma Immunol. 83(5): 413-21.
https://pubmed.ncbi.nlm.nih.gov/10582722

II. Nopp A, Lundahl J, Hallden G (2000). Quantitative, rather than qualitative, differences in CD69 upregulation in human blood eosinophils upon activation with selected stimuli. Allergy. 55(2): 148-56.
https://pubmed.ncbi.nlm.nih.gov/10726729

III. Nopp A, Hallden G, Lundahl J, Johansson E, Vrtala S, Valenta R, Gronneberg R, Van Hage-Hamsten M (2000). Comparison of inflammatory responses to genetically engineered hypoallergenic derivatives of the major birch pollen allergen bet v 1 and to recombinant bet v 1 wild type in skin chamber fluids collected from birch pollen-allergic patients. J Allergy Clin Immunol. 106(1 Pt 1): 101-9.
https://pubmed.ncbi.nlm.nih.gov/10887312

IV. Nopp A, Lundahl J, Stridh H (2002). Caspase activation in the absence of mitochondrial changes in granulocyte apoptosis. Clincal and Experimental Immunology.

V. Nopp A, Stridh H, Gronneberg R, Lundahl J (2002). Differences in neutrophil and eosinophil survival between allergic and non-allergic individuals. [Submitted]