Defense Date

2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Microbiology & Immunology

First Advisor

Daniel Conrad

Abstract

CD23, the natural negative regulator of IgE, has been shown to be involved in asthma progression through its regulation of IgE. To investigate if its sheddase, ADAM10, is also involved in asthma progression, three mouse models were utilized; an IgE/mast cell dependent model, an IgE dependent, mast cell independent model and a mast cell and IgE independent model. Experimental asthma was then induced in mice which were selectively deficient for ADAM10 in B cells (ADAM10-/-) and compared to WT controls. The ADAM-/- mice had decreased signs of asthma, including eosinophilia, AHR and IgE synthesis in the IgE dependent model compared to LM controls, while with the IgE independent model there was no significant difference. Thus, CD23Tg and ADAM10-/- B cell mice have reduced IgE dependent lung inflammation in mouse models compared to WT controls. As a follow up, ADAM10 was inhibited in WT mice by intranasal administration of an ADAM10 inhibitor, compared to carrier (DMSO) treated mice. As with ADAM10-/- mice, inhibition of ADAM10 was only able to control IgE dependent models. These results thus show that ADAM10 is a possible target in controlling IgE dependent allergic disease, possibly as blocking ADAM10 would cause an increase in CD23 membrane expression. To better understand how ADAM10 cleaves CD23 we first sought to confirm previous studies that CD23 is internalized, with the hypothesis that shedding takes place intracellularly, rather than at the cell surface as previously assumed. Indeed, ADAM10 is more highly expressed intracellularly than at the cell surface. At 37 ºC, crosslinking CD23, especially with the anti-stalk mAb 19G5, resulted in extensive CD23 internalization. In addition, the expected increase in soluble CD23 (sCD23) production when 19G5 was added was blocked by the addition of NH4Cl. NH4Cl is known to block the progression of the endosomal pathway. These findings thus confirmed our hypothesis that cleavage of CD23 requires internalization and progression through the endosomal pathway before it is released into the extracellular space. We further demonstrated that ADAM10 is not only involved in cleaving CD23, but also in sorting CD23 into exosomes, as B cells lacking ADAM10 do not incorporate CD23 into exosomes. In addition, we found that exosomes secreted from the cell contain full length CD23, thus showing that they could bind IgE/antigen complex and be involved in the known CD23 dependent enhancement of antigen presentation by the injection of IgE/antigen complexes compared to antigen alone. These results also show that the change in ADAM10 expression specifically in a B cell could be involved in enhancement of IgE dependent inflammation. To determine what signals change ADAM10 expression, ADAM10 promoter studies were initiated. We found that both IL-21 and anti-CD40 increased ADAM10 promoter activity, while IL-4 and IL-13 had no effect. Overall our data show that increasing ADAM10 activity and expression leads to increased inflammation and IgE and is a possible target in controlling IgE dependent diseases.

Rights

© The Author

Is Part Of

VCU University Archives

Is Part Of

VCU Theses and Dissertations

Date of Submission

May 2011

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