The rs1143679 (R77H) lupus associated variant of ITGAM (CD11b) impairs complement receptor 3 mediated functions in human monocytes

Abstract

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 (sRBC(iC3b)) 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 sRBC(iC3b), 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.

Figure 1

Cell-surface expression of CD11b quantified by flow cytometry. Data are presented for neutrophils and monocytes in the resting state and after 10 min stimulation with 200 nM phorbol myristate acetate. Mean fluorescence intensity (MFI) is presented. There are no significant differences between groups. ICRF, anti-CD11b antibodies.

Figure 2

Relative expression of ITGAM mRNA in wild-type and heterozygous volunteers. There are no significant differences between groups. (A) Monocytes. (B) Peripheral blood mononuclear cells (PBMC).

Figure 3

Expression of the CD11b CBRM1/5 activation epitope on phorbol myristate acetate-stimulated monocytes and neutrophils expressed either as a mean fluorescence intensity (MFI) or a percentage of positive cells. There are no significant differences between groups.

Figure 4

Phagocytosis of iC3b opsonised sheep erythrocytes (sRBC_iC3b) by ex-vivo monocyte-derived macrophages (A) and transfected COS7 cells (B). Significant differences in phagocytic index and percentage of phagocytosis are observed, but no significant difference in association index. WT, wild-type.

Figure 5

Adhesion of ex-vivo monocytes to ligand-coated plates. Adhesion of 77H monocytes expressed as a percentage of the wild-type (WT) monocyte adhesion in paired assays. Significant genotype-specific differences in the adhesion to all four CR3 ligands was observed but no difference in adhesion to anti-CD11b (ICRF) antibodies.

Figure 6

The reduction in Toll-like receptor (TLR)7/8-induced cytokine release seen when monocyte CR3 is pre-engaged using iC3b opsonised sheep erythrocytes (sRBC_iC3b). The response of wild-type (WT) (shaded) and 77H cells (unshaded) is shown with p values showing the significant difference in TLR7/8 response between CD11b genotypes. IL, interleukin; TNFα, tumour necrosis factor alpha.