Background/Purpose:

ITGAM encodes CD11b, the α_M subunit of the Mac-1 -α_Mβ₂ integrin. Mac-1 has many functions on leukocytes including its role as an adhesion molecule and complement receptor.  Genome wide association studies (GWAS) have demonstrated that a non-synonymous SNP in the ITGAM locus, rs1143679 encoding an Arg/His in the extracellular domain, is associated with SLE susceptibility. This variant is in strong linkage disequilibrium with another common non-synonymous SNP (rs1143678 encoding a Ser/Lys in the cytoplasmic domain). We have explored possible functional changes due to these ITGAM variants through analysis of expression level, activation and cell function using ex vivo approach with neutrophils from genotyped healthy donors.

Methods:

Neutrophils from healthy control donors genotyped at both rs1142679 and rs1143678 were isolated for ex-vivo functional studies.  The phagocytic potential of CD11b variants was probed with complement coated erythrocytes (EAC) and serum treated zymosan (STZ).  The adhesion potential of ITGAM variants was assessed in a flow chamber analyzing neutrophil adhesion to both ligand (ICAM-1) and endothelial cells.  Total neutrophil CD11b expression and expression of the activation dependent I-domain was assessed by flow cytometry.

Results:

In 2424 healthy control donors, we confirmed strong LD between SNP rs1134678 and rs1143679 (D’=0.97, r=0.82).  We assessed the functional potential of neutrophils from donors heterozygous for each SNP alone using STZ to determine complement dependent binding and phagocytosis.  Variation at either SNP results in a quantitative decrease in STZ phagocytosis and that donors heterozygous for both variants had even lower phagocytosis (repeated measures ANOVA, p<0.04, n=3).  Quantitative complement dependent phagocytosis was also significantly decreased in donors homozygous for the variant alleles (39% decrease for STZ, n=3 pairs, p<0.02/38% decrease for EAC, n=3 pairs, p<0.02).  In a flow chamber based assay of neutrophil adherence, cells from donors homozygous for the variant alleles of rs1134678 and rs1143679 adhered significantly less than neutrophils for donors homozygous for the common alleles (adherence to ICAM-1/endothelial cells:  42% decrease, n=3 pairs, p<0.002/46% decrease, n=4 pairs, p<0.001).

These functional changes in neutrophil Mac-1 function were not attributable to differences in total CD11b expression nor to difference in expression of the activation induced I-domain in donors homozygous for the common vs. variant alleles of ITGAM.

Conclusion:

We demonstrate that ITGAM variants rs1143678 and rs1143679 SNP each make separate contributions to alterations in Mac-1 function on human neutrophils.  These functional alterations are not caused by differences in quantitative total receptor expression or expression of the I-domain.  The study of the functional consequences of allelic variants associated with disease in primary human cells is necessary to fully understand the role of disease associated genetic variants.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/functional-genomics-of-the-human-itgam-locus/