Phagocytosis is the main CR3-mediated function affected by the lupus-associated variant of CD11b in human myeloid cells.

The CD11b/CD18 integrin (complement receptor 3, CR3) is a surface receptor on monocytes, neutrophils, macrophages and dendritic cells that plays a crucial role in several immunological processes including leukocyte extravasation and phagocytosis. The minor allele of a non-synonymous CR3 polymorphism (rs1143679, conversation of arginine to histidine at position 77: R77H) represents one of the strongest genetic risk factor in human systemic lupus erythematosus, with heterozygosity (77R/H) being the most common disease associated genotype. Homozygosity for the 77H allele has been reported to reduce adhesion and phagocytosis in human monocytes and monocyte-derived macrophages, respectively, without affecting surface expression of CD11b. Herein we comprehensively assessed the influence of R77H on different CR3-mediated activities in monocytes, neutrophils, macrophages and dendritic cells. R77H did not alter surface expression of CD11b including its active form in any of these cell types. Using two different iC3b-coated targets we found that the uptake by heterozygous 77R/H macrophages, monocytes and neutrophils was significantly reduced compared to 77R/R cells. Allele-specific transduced immortalized macrophage cell lines demonstrated that the minor allele, 77H, was responsible for the impaired phagocytosis. R77H did not affect neutrophil adhesion, neutrophil transmigration in vivo or Toll-like receptor 7/8-mediated cytokine release by monocytes or dendritic cells with or without CR3 pre-engagement by iC3b-coated targets. Our findings demonstrate that the reduction in CR3-mediated phagocytosis associated with the 77H CD11b variant is not macrophage-restricted but demonstrable in other CR3-expressing professional phagocytic cells. The association between 77H and susceptibility to systemic lupus erythematosus most likely relates to impaired waste disposal, a key component of lupus pathogenesis

Identification and Characterization of a Lupus Suppressor 129 Locus on Chromosome 3

The 129-derived Sle16 is a susceptibility locus for systemic autoimmunity when present on the C57BL/6 (B6) background. Genetic analysis of a (129×B6)F2 cross identified a region from the B6 chromosome 3 (Sle18) with positive linkage to anti-nuclear antibodies. Here we have generated a B6 congenic strain harbouring the 129 allele of Sle18 and intercrossed this line with the lupus-prone B6.129-Sle16 strain. The presence of the 129-Sle18 allele in the B6.129-Sle16Sle18 double congenic mice suppressed the development of Sle16-mediated autoantibody production and ameliorated the renal pathology. The 129-Sle18 locus rectified the B cell abnormalities detected in the B6.129-Sle16 mice, such as the reduction in the percentage of marginal zone B and B1a cells and the increased number of germinal centers. The B6.129-Sle16Sle18 spleens still displayed an increased percentage of activated T and B cells. However, in the B6.129-Sle16Sle18 strain the percentage of naïve T cells was equivalent to that in B6.129-Sle18 and B6 mice and these cells showed a reduced proliferative response to anti-CD3 stimulation compared to B6.129-Sle16 T cells. There was a significant increase in the percentage of CD4(+)FoxP3(+)regulatory T cells in all congenic strains. These cells had normal regulatory function when tested in vitro. Thus 129-Sle18 represents a novel, non-MHC lupus-suppressor locus probably operating as a functional modifier of B cells that, in combination with other factors, leads to lupus resistance. Further characterisation of this locus will help to uncover the immune mechanism(s) conferring protection against lupus

Cytokine response.

<p>Monocytes (A), DCs (B) were stimulated with 2 µg/ml and 10 µg/ml of TLR7/8 ligand (R848) respectively for 24 h. Cytokines quantified using a bead multiplex assay. Closed symbols: 77R/R cells, open symbols: 77R/H-77H/H cells. Each dot represents a single individual, bars denote means. No significant differences between the two CD11b genotypes. Statistical analysis by paired t test. (C, D) Modulation of TLR7/8-induced cytokine release by hiC3b-coated beads. Monocytes (C), DCs (D) were fed with hiC3b-coated beads one hour prior to R848 stimulation. The cytokine changes between the samples with and without CR3 pre-engagement with iC3b are shown with the p values indicated. Data are expressed as mean+/−SEM. The cytokine responses of 77R/R cells (black column) and 77R/H-77H/H cells (white columns) were not statistically different in paired assays. IL, interleukin; TNF-α, tumour necrosis factor alpha; IP-10, Interferon gamma-induced protein 10.</p

<i>In vitro</i> and <i>in vivo</i> PMN migration.

<p>(A) Migration of neutrophil cell lines through transwells seeded with mouse endothelial cells. PMNs migrated into the bottom chamber in response to MIP-2 were counted at different time points as indicated. Pooled results from at least 4 independent experiments are presented as mean ± SEM. <i>Itgam</i>−/− and wild type C57BL/6 neutrophil cell lines were used as controls. CD11b-deficient PMNs, known to have weaker endothelial interactions, migrated faster than the C57BL/6 and the hCD11b expressing cell lines (p<0.001 at 60 mns and p<0.05 at 90 mns). Statistical analysis by Bonferroni's multiple comparison test. (B) Time course of the migration of freshly isolated human 77R/R or 77R/H neutrophils through a HUVEC layer in response to MIP-2. Pooled results from at least 4 independent experiments are presented as mean ± SEM. (C, D) In vivo peritoneal migration of hCD11b-77R and hCD11b-77H PMN cells lines following i.p. injection of MIP-2 (C) and thioglycollate (D). The two hCD11b expressing PMN lines were labelled with DDAO or CFSE and adoptive transferred at a 1∶1 ratio into C57BL/6 mice. Absolute numbers of labelled PMNs recovered from the peritoneum are shown. Data of one out of at least 3 independent experiments are presented. Bars indicate means.</p

Phagocytosis of hiC3b-coated fluorescent beads.

<p>The uptake by macrophages (A), PMNs (B), monocytes (C) and DCs (D) was quantified by flow cytometry and the data are represented as percentage of phagocytosis. Data are expressed as mean+/−SEM. Filled columns 77R/R individuals, open columns 77R/H-77H/H individuals. Paired T test was applied and the p values are indicated.</p

Cell surface expression of CD11b on different cell populations.

<p>The expression was quantified by flow cytometry using ICRF44 (A) and CBRM1/5 (B) antibodies. The latter only recognises the headpiece of CD11b in its active state. Data are presented in mean fluorescence intensity (MFI), closed symbols 77R/R donors, open symbols 77R/H-77H/H donors. The two groups were not statistically different. Bars indicate means.</p

Rosetting assay.

<p>Percentage of rosettes formed by CFSE-labelled RBC-hiC3b with freshly isolated PMNs from donors carrying the susceptible allele (77H/H, open symbol) or the common allele (77R/R, closed symbol). One representative assay out of 3 independent experiments.</p