Neutrophil transmigration mediated by the neutrophil-specific antigen CD177 is influenced by the endothelial S536N dimorphism of platelet endothelial cell adhesion molecule-1

Abstract

The human neutrophil-specific adhesion molecule CD177 (also known as the NB1 alloantigen) becomes upregulated on the cell surface in a number of inflammatory settings. We recently showed that CD177 functions as a novel heterophilic counterreceptor for the endothelial junctional protein PECAM-1 (CD31), an interaction that is mediated by membrane-proximal PECAM-1 IgD 6, which is known to harbor an S(536)N single nucleotide polymorphism of two major isoforms V(98)N(536)G(643) and L(98)S(536)R(643) and a yet-to-be-determined region on CD177. In vitro transendothelial migration experiments revealed that CD177(+) neutrophils migrated significantly faster through HUVECs expressing the LSR, compared with the VNG, allelic variant of PECAM-1 and that this correlated with the decreased ability of anti-PECAM-1 Ab of ITIM tyrosine phosphorylation in HUVECs expressing the LSR allelic variant relative to the VNG allelic variant. Moreover, engagement of PECAM-1 with rCD177-Fc (to mimic heterophilic CD177 binding) suppressed Ab-induced tyrosine phosphorylation to a greater extent in cells expressing the LSR isoform compared with the VNG isoform, with a corresponding increased higher level of beta-catenin phosphorylation. These data suggest that heterophilic PECAM-1/CD177 interactions affect the phosphorylation state of PECAM-1 and endothelial cell junctional integrity in such a way as to facilitate neutrophil transmigration in a previously unrecognized allele-specific manner.

Figure 1

Nucleotide sequencing analysis of polymorphic PECAM-1 from three different HUVECs. mRNA extracted from HUVECs were reverse transcribed into cDNA and amplified with PECAM-1–specific primers and sequenced. Arrows indicate the position of SNP of PECAM-1 encoding asparagine/serine dimorphism (underline) at position 536 located in IgD6.

Figure 2

A, FACS analysis of adhesion molecules on SS (white) and NN (black) phenotyped HUVECs. HUVECs in resting (−TNF-α) or stimulated conditions (+TNF-α) were labeled with mAbs against PECAM-1, JAM-C, ICAM-1, E-Selectin, and mIgG (as control). Bound Abs were detected by FITC-labeled secondary Ab and were analyzed by flow cytometry. B, Permeability of three phenotyped HUVEC lines. HUVECs were cultured on fibronectin-coated polycarbonate filter chambers for 48 h. The passage of FITC-albumin through a confluent monolayer of cells at different time periods (5–60 min) was measured in the presence and absence of 0.2 U/ml thrombin or 3.2 × 10⁻⁵ mol/l histamine (upper panel). The lower panel shows permeability after treatment with mAb PECAM-1.1 in the presence and absence of rCD177/Fc or rJAM-C/Fc (as control). The intensity of migrated FITC-albumin in the lower chamber was measured by fluorescence microtiter plate reader. *p < 0.01; FITC-albumin passage between homozygous SS and NN (n = 4). AU, arbitrary unit.

Figure 3

Analysis of CD177 and PECAM-1 surface expression in neutrophils by FACS. Neutrophils of CD177⁻ (left panel) and CD177⁺ (right panel) individuals were labeled with mAb 7D8 or mAb Gi18. After washings, bound Abs were detected by FITC-labeled secondary Ab and were analyzed by flow cytometry.

Figure 4

A, Analysis of neutrophil transmigration through PECAM-1–phenotyped HUVECs. HUVECs were cultured on fibronectin-coated polycarbonate membranes in transmigration chambers. After 48 h, fluorescence-labeled neutrophils (CD177⁺ or CD177⁻; see Fig. 3) were allowed to transmigrate through HUVECs for 90 min toward fMLP (10⁻⁸ M) or without fMLP (as control). Fluorescence intensity of migrated neutrophils in the lower chamber was measured by fluorescent microtiter plate reader (n = 4). B, Inhibition of neutrophil migration through PECAM-1–phenotyped HUVEC mAb PECAM-1 against IgD6 (PECAM-1.2) and IgD1 (PECAM-1.3). Fluorescence-labeled CD177⁺ neutrophils were allowed to pass through untreated and treated HUVECs toward fMLP and were measured as above (n = 4). C, Analysis of neutrophil transmigration through PECAM-1–phenotyped HUVECs with different chemoattractants. Fluorescence-labeled neutrophils (CD177⁺) were allowed to transmigrate through HUVECs for 90 min toward fMLP (10⁻⁸M), IL-8 (50 ng/ml), or LTB-4 (10⁻⁴ M) or without chemoattractant (as control) and were measured as above. *p < 0.05; **p < 0.01; neutrophil migration between homozygous SS and two NS and NN phenotypes (n = 4).

Figure 5

A, Analysis of CD177 and PECAM-1 surface expression on phenotyped neutrophils (CD177⁺ or CD177⁻ neutrophils) before and after treatment with PLY. Neutrophils were treated with 25 ng/ml PLY for 30 min and labeled with mAbs 7D8 (anti-CD177) or Gi18 (anti–PECAM-1). After washing, bound mAb was detected with FITC-labeled secondary Ab and analyzed by FACS. B, PLY-untreated (−) or -treated (+) fluorescence-labeled neutrophils were allowed to migrate through HUVECs toward fMLP and were measured as above. *p < 0.05; **p < 0.01; neutrophil migration between homozygous SS and two NS and NN phenotypes (n = 4).

Figure 6

Immunoblot analysis of PECAM-1 phosphorylation in NN- or SS-phenotyped HUVECs after different stimulation. A, HUVECs were stimulated with rCD177/Fc, mAb PECAM-1.1 or PECAM-1.2 or PECAM-1.3 and PECAM-1.1 followed by rCD177/Fc (2.5 μg/ml for 60 min). After lysis, aliquots of protein (5 μg) were run on 10% SDS-PAGE. After blotting, proteins were stained with anti–PECAM-1 pY686 Ab (1 μg/ml), mAb Gi18 against PECAM-1 (2.5 μg/ml), or mAb against GAPDH (2.5 μg/ml) and were visualized with HRP-labeled secondary Abs using a chemiluminescence system. B, HUVECs were stimulated with H₂O₂ in the presence of rCD177/Fc or Fc alone (2.5 μg/ml for 60 min). After blotting, proteins were visualized with anti–PECAM-1 pY686 Ab (1 μg/ml), mAb Gi18 against PECAM-1 (2.5 μg/ml), anti–β-catenin pT41/S45 (2.5 μg/ml), or GAPDH (2.5 μg/ml), as described above. Bar graphs (right panels) represent the ratio of phosphorylated PECAM-1 (p– PECAM-1) or phosphorylated β-catenin (p–β-catenin) to total PECAM-1. *p < 0.01; PECAM-1 phosphorylation level between homozygous SS and NN.

Figure 7

Effect of the PECAM-1 allelic form on ligand binding. Binding of mAb PECAM-1.2 and CD177 with the SS and NN allelic isoforms of PECAM-1 protein was determined in real time using SPR. Purified PECAM-1 isoforms were immobilized on a sensor chip. MAb PECAM-1.2 (upper panels) or purified CD177 (lower panels) was injected with a flow rate of 100 or 20 μl/ml, respectively, at 25°C. Note that CD177 bound with similar affinity to either PECAM-1 isoform as did mAb PECAM-1.2.