No association between neutrophil FcgammaRIIa allelic polymorphism and anti-neutrophil cytoplasmic antibody (ANCA)-positive systemic vasculitis

Abstract

ANCA, implicated as having a pathogenic role in systemic vasculitis, can activate tumour necrosis factor-alpha (TNF-alpha)-primed neutrophils by cross-linking surface-expressed ANCA antigens with neutrophil FcgammaRIIa receptors to release reactive oxygen species. The FcgammaRIIa receptor exists as polymorphic variants, R131 and H131, which differ in their ability to ligate human IgG2 and IgG3. Neutrophils homozygous for the FcgammaRIIa-H131 allotype bind more efficiently to IgG3 than the FcgammaRIIa-R131 allotype and are the only human FcgammaR which bind IgG2. Our aim was to determine whether the homozygous FcgammaRIIa-H131 individuals are more susceptible to developing ANCA-associated systemic vasculitis and nephritis due to differential IgG binding and activation. FcgammaRIIa allotype was determined by both allele-specific polymerase chain reaction (PCR) and Southern blotting with allele-specific oligonucleotide probes end-labelled with 32P-gammaATP, after PCR amplification of genomic FcgammaRIIa DNA in 107 Caucasian patients with ANCA+ vasculitis (of whom 89 had renal disease) and 100 ethnically matched controls. Phenotyping of neutrophil FcgammaRIIa alleles was confirmed in some patients by quantitative flow cytometry using murine MoAbs 41H16 and IV.3. Of the patients with ANCA+ systemic vasculitis, 75 had ANCA with specificity for proteinase 3 and 32 with specificity for myeloperoxidase. Overall, no skewing in FcgammaRIIa allotypes was seen in patients compared with controls. No significant increase of the FcgammaRIIa-H131 allotype was found amongst patients irrespective of ANCA specificity, and no association between the FcgammaRIIa allotype and nephritis was found. Our data suggest that the FcgammaRIIa receptor allotype is not a major factor predisposing to the development of ANCA+ systemic vasculitis, or to nephritis.

Fig. 1

Agarose gel electrophoresis of amplified DNA by polymerase chain reaction (PCR) using allele-specific primers for FcγRIIa-H/H131 and R/R131, as described in Subjects and Methods. The FcγRIIa-specific primers amplify a 290-bp product. Amplified DNA from nine individuals (three H/H131, three R/H131, and three R/R131) and two cell lines (K562, R/H131 and U937, R/R131). K, K562; U, U937.

Fig. 2

Superimposed Southern blots of amplified DNA hybridized with ³²P-γATP-labelled FcγRIIa allele-specific oligonucleotide probes (H/H131 and R/R131), as described in Subjects and Methods. Amplified DNA from nine individuals (three H/H131, three R/H131, and three R/R131) and two cell lines (K562, R/H131 and U937, R/R131). Neg, Negative; K, K562; U, U937.

Fig. 3

Expression of FcγRIIa receptors on human neutrophils with the (a) R/R131, (b) R/H131, and (c) H/H131 phenotypes. Each profile represents 10 000 neutrophils, stained with murine MoAbs IV.3 (binds both FcγRIIa alleles) and 41H16 (binds FcγRIIa-R131 only).