The Regulation of Neutrophil Extracellular Trap-induced Tissue Damage by Human CD177

Abstract

Background: Neutrophil-induced tissue damage contributes to the rejection in xenotransplantation. Therefore, suppressing neutrophil function could be effective in suppressing xenogeneic rejection. In a previous study, we demonstrated that the ectopic expression of human cluster of differentiation 31 (CD31) on porcine endothelial cells (PEC) significantly suppressed neutrophil-mediated cytotoxicity through the homophilic binding of CD31. Cluster of differentiation 177 (CD177) was recently reported to be a high-affinity heterophilic binding partner for CD31 on endothelial cells. Thus, we hypothesized that human CD177 on PEC might induce a stronger suppression in neutrophil-mediated cytotoxicity compared with CD31. In this study, the inhibitory function of human CD177 on PEC in neutrophil-mediated cytotoxicity was investigated.

Methods: PEC were transfected with a cloning plasmid containing cDNA inserts that encoded for hCD177 and hCD31 genes. Neutrophil-induced cytotoxicity was evaluated by flow cytometry after coculturing with PEC or PEC/CD177 in the presence of phorbol 12-myristate 13-acetate. To elucidate the mechanisms responsible for hCD177-induced suppression, the phosphorylation of src homology region 2 domain containing phosphatase 1 was measured by immunoblot analysis.

Results: Human CD177 on PEC induced a significant reduction in neutrophil-induced cytotoxicity. In addition, CD177 on PEC induced a significant increase in the phosphorylation of src homology region 2 domain-containing phosphatase 1 in neutrophils and suppressed NETosis.

Conclusions: These findings suggest that human CD177 suppresses neutrophil-mediated cytotoxicity through the inhibition of NETosis.

FIGURE 1.

Successful transfection of human CD177 and CD31 in PEC. Expression of hCD31, the inhibitory receptor for CD177, in human neutrophils was confirmed by flow cytometry (A). To obtain PEC transfectants (PEC/hCD177 and PEC/hCD31), plasmids containing human CD177 and CD31 were transfected into PEC. The expression of human CD177 and CD31 in the obtained clones was evaluated by flow cytometry. The expression of human CD177 and CD31 in the obtained clones was evaluated by flow cytometry. More than 95% positive staining was observed in PEC/hCD177 (C) and PEC/hCD31 (D), and no expression was detected in naive PEC (B). CD177, cluster of differentiation 177; CD31, cluster of differentiation 31; PEC, porcine endothelial cells.

FIGURE 2.

hCD177-induced suppression of neutrophil-mediated cytotoxicity. Neutrophils were isolated, and the purity was checked by CD11b staining. PEC, PEC/hCD177, and PEC/hCD31 were plated at a concentration of 6 × 10⁴ cells/well as target cells in a 24-well culture plate. After culturing for 24 h, PEC and PEC transfectants were cocultured with 3 × 10⁵ neutrophils for 2 h in the presence of 200 nmol/L PMA, followed by triple-color staining with Annexin V-FITC, 7-AAD, and APC-conjugated anti-CD11b antibody. CD11b-negative cells were gated as the target cells (P1) (A). The % cytotoxicity was calculated as (% Annexin V⁺ 7-AAD⁺ cells) + (% Annexin V⁺ 7-AAD^– cells) + (% Annexin V^– 7-AAD⁺ cells) (B,C). The bars indicate the SEM, *P < 0.01, **P < 0.001, vs PEC. APC, allophycocyanin; FITC, fluorescein isothiocyanate; N.S., no significant difference, n = 7; PMA, phorbol 12-myristate 13-acetate; PEC, porcine endothelial cells.

FIGURE 3.

hCD177-induced suppression in neutrophil is mediated at least partially by the binding of CD177 to CD31. PEC and PEC/hCD177 were plated at a concentration of 6 × 10⁴ cells/well as target cells in a 24 well gelatin-coated plate. After culturing for 24 h, 3 × 10⁵ neutrophils were added to each well. Neutrophils were treated for 1 h with vehicle (PEC, PEC/hCD177) or 200 μg/mL of anti-human CD31 (α CD31) or isotype mouse IgG (Iso). Cells were cocultured for 2 h in the presence of 200 nmol/L PMA, followed by triple-color staining with Annexin V-FITC, 7-AAD, and APC-conjugated anti CD11b antibody. The % cytotoxicity was calculated as (% Annexin V⁺ 7-AAD⁺ cells) + (% Annexin V⁺ 7-AAD^– cells) + (% Annexin V^– 7-AAD⁺ cells). The bars indicate the SEM. *P < 0.05, **P < 0.005. APC, allophycocyanin; CD177, cluster of differentiation 177; CD31, cluster of differentiation 31; FITC, fluorescein isothiocyanate; N.S., no significant difference, n = 8; PEC, porcine endothelial cells; PMA, phorbol 12-myristate 13-acetate.

FIGURE 4.

Suppression of NETosis by human CD177. PEC and PEC/hCD177 were cultured in 24 well gelatin-coated plates at a concentration of 1.6 × 10⁵ cells/well. After 24 h culturing, PEC and PEC transfectants were cocultured with 3 × 10⁵ neutrophils for 2 h in the presence of 50 nmol/L PMA. The cells were procured and stained with PE-labeled anti-CD66b and 50 nmol/L SYTOX green. Neutrophils were gated in CD66b positive, as shown in the black box. Representative data are demonstrated in (A, B), and a significantly decreased %NETosis was detected in the neutrophils that were cocultured with PEC/hCD177 (C). A significant decrease of SYTOX Green positive dead cells was detected in CD66b positive neutrophils that were cocultured with PEC/hCD177 compared with neutrophils with PEC. The bars indicate the SEM, *P < 0.05, vs PEC, n = 5. PE, phycoerythrin; PEC, porcine endothelial cells; PMA, phorbol 12-myristate 13-acetate.

FIGURE 5.

SHP-1 phosphorylation induced by hCD177 and hCD31. PEC, PEC/hCD177, and PEC/hCD31 were seeded in gelatin-coated 6 well plates at a concentration of 1.6 × 10⁵ cells/well and cocultured with neutrophils at a concentration of 1.6 × 10⁶/well. Proteins were extracted from neutrophils after 30 min coculturing in the presence of 200 nmol/L PMA and loaded into 10% SDS-PAGE gel, followed by the transfer to PVDF membranes. The blot was stained with antiphosphorylated SHP-1 mAb and HRP-coupled secondary antibody. Representative data for 7 experiments are shown in (A). The extent of phosphorylation of SHP-1 by hCD177 and hCD31 was quantitatively evaluated using the Image J software program. All results of relative phosphorylated SHP-1 values are shown as the mean ± SEM (B). *P < 0.05. HRP, horseradish peroxidase; N.S., no significant difference, n = 7; PEC, porcine endothelial cells; PMA, phorbol 12-myristate 13-acetate; PVDF, polyvinylidene difluoride; SHP-1, src homology region 2 domain-containing phosphatase 1.