Developing methods for the identification and isolation of dendritic cells from peripheral blood and their application to the study of high affinity IgE receptor (FcεRI) expression by dendritic cells in health and atopic asthma

Hartley, Judith Ann (1999) Developing methods for the identification and isolation of dendritic cells from peripheral blood and their application to the study of high affinity IgE receptor (FcεRI) expression by dendritic cells in health and atopic asthma. University of Southampton, Doctoral Thesis.

Abstract

The aim of this work was to determine whether the number of FcεRI positive dendritic cells and the levels of expression and occupancy of this receptor by IgE were increase in atopic asthmatics compared to normal subjects.

A method has been successfully developed to identify peripheral blood dendritic cells by flow cytometry as cells staining brightly for HLA-DR but negative for a cocktail of lineage specific antibodies (CD3, CD14, CD19, CD16 and CD56).

PBMCs were purified from 35 subjects who had been characterised as to their atopic and asthmatic status. Three-colour flow cytometry was used to determine the levels of expression of FcεRI and CD23 by blood dendritic cells, and their ability to bind IgE in vitro. Statistical analysis revealed that the total expression of FcεRI on the surface of dendritic cells from normal and asthmatic subjects was not significantly different. However, dendritic cells from asthmatics had higher levels of IgE bound to the cell surface in vivo, and could further bind more IgE in vitro than dendritic cells from normal subjects. Dendritic cells were not found to express significant levels of CD23. The subunit composition of the receptor was resolved by RT-PCR on purified dendritic cells, and showed that in the peripheral blood, mRNA transcripts were present for the FcεRI-α, -β and -γ subunits.

In conclusion, total levels of FcεRI expression by dendritic cells in the blood from normal and asthmatic subjects were not different, in marked contrast to receptor expression by dendritic cells in the lung. It is possible therefore that the local environment of the asthmatic lung leads to this upregulation of surface FcεRI. It does appear that the function of the receptor, and in particular its capacity to bind IgE, may be different in health and disease. An improved understanding of the expression of FcεRI by dendritic cells in health and disease may open new avenues for therapy to combat the suffering caused by asthma.

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