Objectives The rs1143679 variant of ITGAM, encoding the R77H variant of CD11b (part of complement receptor 3; CR3), is among the strongest genetic susceptibility effects in human systemic lupus erythematosus (SLE). The authors aimed to demonstrate R77H function in ex-vivo human cells.
Methods Monocytes/monocyte-derived macrophages from healthy volunteers homozygous for either wild type (WT) or 77H CD11b were studied. The genotype-specific expression of CD11b, and CD11b activation using conformation-specific antibodies were measured. Genotype-specific differences in iC3b-mediated phagocytosis, adhesion to a range of ligands and the secretion of cytokines following CR3 ligation were studied. The functionality of R77H was confirmed by replicating findings in COS7 cells expressing variant-specific CD11b.
Results No genotype-specific difference in CD11b expression or in the expression of CD11b activation epitopes was observed. A 31% reduction was observed in the phagocytosis of iC3b opsonised sheep erythrocytes (sRBCiC3b) by 77H cells (p=0.003) and reduced adhesion to a range of ligands: notably a 24% reduction in adhesion to iC3b (p=0.014). In transfected COS7 cells, a 42% reduction was observed in phagocytosis by CD11b (77H)-expressing cells (p=0.004). A significant inhibition was seen in the release of Toll-like receptor 7/8-induced pro-inflammatory cytokines from WT monocytes when CR3 was pre-engaged using sRBCiC3b, but no inhibition in 77H monocytes resulting in a significant difference between genotypes (interleukin (IL)-1β p=0.030; IL-6 p=0.029; tumour necrosis factor alpha p=0.027).
Conclusions The R77H variant impairs a broad range of CR3 effector functions in human monocytes. This study discusses how perturbation of this pathway may predispose to SLE.
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Funding The Twins UK National Institute for Health Research (NIHR) bioresource is funded by the Wellcome Trust, Europeans Community's Seventh Framework Programme (FP7/2007-2013) and ENGAGE project grant agreement (HEALTH-F4-2007-201413). The Department of Health via the NIHR comprehensive Biomedical Research Centre award to Guy's and St Thomas' NHS Foundation Trust in partnership with King's College London provided support for the Twins UK cohort and funding for flow cytometry. Original genotyping was performed by the Wellcome Trust Sanger Institute, in support of the National Eye Institute via an NIH/CIDR genotyping project. All other costs were met by an Arthritis Research UK clinician scientist fellowship (18544) awarded to BR, a fellowship within the post-doc programme of the German Academic Exchange Service (DAAD) and grant J20 of the Interdisciplinary Center for Clinical Research (IZKF) at the University Hospital of the University of Erlangen-Nuremberg awarded to BGF, and the IMI-funded project ‘Be the Cure’. TS is an NIHR senior investigator and is holder of an ERC advanced principal investigator award.
Ethics approval The study was approved by the South East London Research Ethics Committee. Additional volunteers were recruited at the University of Erlangen-Nuremberg, with approval from the ethics committee of the Friedrich Alexander University of Erlangen-Nuremberg.
Patient consent Obtained.
Competing interests None.
Provenance and peer review Not commissioned; externally peer reviewed.