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Integrin-ligand interactions and cytokine production by monocytic cells

Edkins, Adrienne Lesley (2008) Integrin-ligand interactions and cytokine production by monocytic cells. PhD thesis, University of Glasgow.

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Abstract

CD23 is a Type II glycoprotein that exists in both membrane-bound (mbCD23) and soluble (sCD23) forms and functions as the low affinity receptor for IgE. CD23 interacts with a range of proteins to fulfil its function in vivo. Through interactions with its ligands IgE and CD21, CD23 regulates the levels of IgE in serum. Soluble CD23 also interacts with members of the integrin family of membrane receptors. Integrins are heterodimeric transmembrane receptors that participate in bidirectional signalling across membranes and have a critical role in cellular adhesion reactions. CD23 interacts with four integrins, αVβ3 and αVβ5 from the αV integrin family, and αMβ2 and αXβ2, from the β2 family of integrins. Using peptide array technology, we identified set of overlapping peptides derived from the soluble CD23 sequence that interact with integrins expressed on the surface of monocytic cells and with purified αVβ3 and αVβ5 integrins in in vitro Biacore assays. These peptides all contained a common basic tripeptide motif, termed the Arg-Lys-Cys (RKC) motif. Integrins traditionally recognise and bind the Arg-Gly-Asp (RGD) tripeptide in their ligands in a cation-dependent manner. However, the in vitro interaction between RKC-containing peptides and purified integrins was determined to be cation-independent, salt-sensitive and independent of RGD binding. The interaction was blocked by full length CD23. Substitution of the residues in the RKC motif reduced or abolished binding of the peptides to integrins expressed on cells and in vitro, as measured by Biacore analysis, and abolished the competition with CD23. Taken together, these data suggest that the RKC motif is the site in CD23 that is recognised and bound by αVβ3 and αVβ5 integrins. The RKC motif can be considered a novel recognition motif for integrins, as it is cation-independent, and its binding is not blocked by the presence of RGD-containing integrin ligands. Therefore it is likely that the RKC motif interacts with integrins at a site other than that used for RGD-binding, similar to the interactions that have been described for the binding of the HIV Tat protein. The interaction between sCD23 and its integrin receptors is important in the regulation of cytokine production by monocytic cells. Most monocytic cells will express a combination of the different CD23-binding integrins simultaneously and, therefore, the cytokine output of that cell will be the net result of the interaction of CD23 with a combination of integrins. We used monoclonal antibodies to investigate the role of individual integrins in cytokine production. Antibodies were selected to allow comparision of the cytokine response between, a) integrin families, b) integrin subunits, and c) integrin epitopes. The cytokine profile induced by integrin ligation did not differ between the αV and β2 integrin families, although the concentration of cytokines produced varied depending on the heterodimer targeted. However, within a particular family, cytokine production induced by integrin ligation was specific and relied on the recognition of a precise epitope on the integrin. Cytokine production by CD23-binding integrins appears to require the ligation of the α subunit of the integrin heterodimer. We identified an antibody directed against the αVβ3 integrin that induced high levels of cytokine production. Cytokine production following ligation of the integrin with this antibody was dependent on activation of the ERK/MAPK pathway in cells. This production of cytokines and phosphorylation of ERK was enhanced by the addition of macrophage colony stimulating factor (M-CSF) and partially inhibited by an anti-TLR-2 antibody. Chronic stimulation (<3 days) of THP-1 cells with the anti-αVβ3 antibody in the presence of M-CSF led to morphological changes in the cell line associated with the development of a more macrophage-like phenotype and the continued production of cytokines. Analysis of the changes in cell surface marker expression and cytokine profiles suggested that the THP-1 cells had undergone an M2b programme of macrophage activation in response to αVβ3 ligation. Data presented herein reinforce the importance of the role of integrins in the control of adhesion-independent signalling pathways in suspension cells.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: CD23, integrin, monocyte, macrophage, inflammation
Subjects: Q Science > Q Science (General)
R Medicine > R Medicine (General)
Colleges/Schools: College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Supervisor's Name: Cushley, Professor W.
Date of Award: 2008
Depositing User: Miss Adrienne L Edkins
Unique ID: glathesis:2008-286
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 01 Dec 2008
Last Modified: 10 Dec 2012 13:17
URI: http://theses.gla.ac.uk/id/eprint/286

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