The human neutrophil-specific adhesion antigen CD177 (NB1, HNA-2a or PRV-1) becomes upregulated on the cell surface in a number of inflammatory settings, and human alloantibodies specific for CD177 have been implicated in the development of transfusion related acute lung injury (TRALI). Recent studies have shown that CD177 functions as an important counter-receptor for the endothelial cell-cell junctional protein PECAM-1 (CD31) – a heterophilic adhesive interaction that appears to be mediated by Ig domain 6 of PECAM-1 and a still-to-be-identified site on CD177. Interestingly, a common single nucleotide polymorphism (SNP) exists within the PECAM-1 gene that produces an S536N amino acid substitution within Ig domain 6 that is proximal to the CD177 binding site. Whether this polymorphism influences PECAM-1/CD177 interactions and subsequent neutrophil transendothelial cell migration, however, is not known. To investigate the potential for the S536N PECAM-1 SNP to affect functionally important interactions between PECAM-1 and CD177, recombinant CD177 and PECAM-1 S536 or N536 allelic isoforms were produced in CHO mammalian cells, purified by affinity chromatography, and subjected to surface plasmon resonance analysis. We found that the S536 isoform of PECAM-1 bound with much higher affinity to CD177 than did its N536 counterpart (Ka for PECAM-1S536/CD177 = 5×10−7 versus a Ka for PECAM-1N536/CD177=1×10−7). To analyze the possible functional impact of the S536N polymorphism on neutrophil transendothelial migration, fMLP-stimulated neutrophils were allowed to transmigrate across human umbilical vein endothelial cell (HUVEC) monolayers that had been genotyped to be either PECAM-1 homozygous SS, heterozygous NS, or homozygous NN. We found that neutrophils migrated significantly faster across HUVECs expressing the PECAM-1S536, rather than the PECAM-1N536, allelic isoform. In addition, western blot analysis using a phospho-specific antibody revealed that cytoplasmic PECAM-1 ITIM tyrosines became phosphorylated to a greater extent in PECAM-1S536- versus PECAM-1N536-expressing HUVECs that had been incubated with either soluble recombinant CD177 or an anti-PECAM-1 mAb specific for Ig domain 6. Taken together, these data demonstrate that
heterophilic interactions between neutrophil CD177 and endothelial cell PECAM-1 play an important role in neutrophil transendothelial migration, and
the naturally-occurring S536N polymorphism within Ig domain 6 likely affects ligand binding-induced downstream PECAM-1 signaling.
Allele-specific, PECAM-1-mediated effects on neutrophil transendothelial migration and inflammatory events such as TRALI may represent an important area for future investigation.
Disclosures: No relevant conflicts of interest to declare.