Variation at ITGAM (CD11b – the alpha chain of complement receptor 3 (CR3)) is among the strongest genetic susceptibility effects identified in human lupus, with rs1143679, encoding an R77H amino acid change, implicated by genetic analysis as the functional variant.
We studied COS7 cells transiently transfected with wild-type (WT) and 77H variant ITGAM alongside the invariant beta chain to reconstitute CR3. Transfected cells were used for a static adhesion assay in 96-well plates coated with various protein ligands and for a phagocytic assay using iC3b opsonised sheep erythrocytes (sRBC). Assays were replicated using ex-vivo monocytes (adhesion) and monocyte-derived macrophages (phagocytosis) from healthy genotyped volunteers.
Phagocytosis of iC3b coated sRBC by COS cells was significantly impaired for cells expressing the 77H mutant compared with WT (mean at 30 minutes 0.48 sRBC/phagocyte vs. 0.87sRBC/phagocyte, P<0.001). The adhesion of sRBCs to the transfected cells was not impaired. Transfected cells bound specifically to 96-well plates coated with known CR3 ligands, most strongly iC3b and DC-SIGN in this static adhesion assay. However, the binding of the 77H transfects to protein coated wells was 39% lower than WT transfects to iC3b and 19% lower to DC-SIGN. Only 1% of the healthy European population are homozygous for the rs1143679, making the recruitment of healthy human participants for study difficult. Nonetheless we also have preliminary data in ex-vivo monocytes that demonstrates functional differences in adhesion and phagocytosis consistent with those found in our cell line model.
We demonstrate functional consequences of the R77H variation in a cell line model and extend this to provide preliminary evidence that these effects are also observed in ex-vivo myeloid cells from healthy individuals. Although further work is required it is apparent that the functional consequences of the rs1143679 mutation may fit within existing theories of lupus pathogenesis that include complement mediated clearance of apoptotic cells and immune complexes.
Disclosure: B. Rhodes, None; B. G. Fürnrohr, None; T. J. Vyse, None.