All ETDs from UAB

Advisory Committee Chair

Jeffrey C Edberg

Advisory Committee Members

Susan L Bellis

Daniel C Bullard

Dennis F Kucik

Alexander J Szalai

Document Type

Dissertation

Date of Award

2014

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Multiple studies have demonstrated that single-nucleotide polymorphisms (SNPs) in the ITGAM locus (including the non-synonymous SNPs rs1143679, rs1143678 and rs1143683) are associated with SLE. ITGAM encodes the protein CD11b, a subunit of the ß2 integrin Mac-1. The purpose of this work was to determine the effects of ITGAM genetic variation on the biological functions of neutrophil Mac-1. Neutrophils from ITGAM genotyped and sequenced healthy donors were isolated for functional studies. The phagocytic capacity of neutrophil ITGAM variants was probed with complement coated erythrocytes, serum treated zymosan, heat treated zymosan and IgG coated erythrocytes. The adhesion capacity of ITGAM variants, in adhering to either purified intercellular adhesion molecule 1 or tumor necrosis factor -α stimulated endothelial cells was assessed in a flow chamber. Expression levels of total CD11b and activation of CD11b were assessed by flow cytometry. Neutrophil extracellular trap release in response to different stimuli was quantitatively analyzed with fluorescent microscopy. Mac-1-mediated neutrophil phagocytosis was significantly reduced in individuals with nonsynonymous variant alleles of ITGAM. This reduction in phagocytosis was related to variation at either rs1143679 (in the ß-propeller region of CD11b) or rs1143678/rs1143683 (highly linked SNPs in the cytoplasmic/calf-1 regions). Phagocytosis mediated by Fcgamma receptors was also significantly reduced in donors with variant ITGAM alleles. Similarly, firm adhesion of neutrophils was significantly reduced in individuals with variant ITGAM alleles. These functional alterations were not attributable to differences in total CD11b receptor expression or activation. Neutrophil extracellular traps releasing caused by serum treated zymosan, heat aggregated IgG and TLR7/8 stimuli - Resiquimod (R848) was reduced in donors with variant ITGAM alleles. However, PMA induced neutrophil extracellular traps releasing and oxidative burst mediated by NADPH oxidase were not altered by ITGAM genetic variation. We showed that both the nonsynonymous ITGAM variants rs1143679 and rs1143678/rs113683 contribute to altered Mac-1 function on neutrophils independently. These results underscore the need to consider multiple nonsynonymous SNPs when assessing the functional consequences of ITGAM variation on immune cell processes and the contribution of ITGAM variants to the risk of SLE.

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