The secreted Ly6/uPAR-related protein-1 suppresses neutrophil binding, chemotaxis, and transmigration through human umbilical vein endothelial cells

Abstract

The secreted Ly-6/uPAR Related Protein-1 (SLURP1) is an immunomodulatory protein that promotes corneal immune- and angiogenic-privilege. Here, we have examined the influence of SLURP1 on neutrophil-vascular endothelial cell interactions using human umbilical vein endothelial cells (HUVEC) and differentiated neutrophil-like HL-60 (dHL-60) cells, or primary human neutrophils. SLURP1 blocked the tumor necrosis factor-alpha (TNF-α)-activated dHL-60 cells (i) binding to TNF-α-activated HUVEC with a concurrent reduction in endothelial cell adhesion molecule E-selectin, (ii) transmigration through TNF-α-activated confluent HUVEC monolayer by stabilizing VE-cadherin and β-catenin on endothelial cell cytoplasmic membranes, (iii) chemotaxis towards chemoattractant formyl Met-Leu-Phe (fMLP) coupled with their decreased polarization, and (iv) TNF-α-stimulated matrix metalloproteinase-9 (MMP9) expression and activity. SLURP1 also suppressed the primary human neutrophil chemotaxis, and interaction with HUVEC. Furthermore, SLURP1 suppressed fMLP-induced phosphorylation of protein kinase-B (AKT) in dHL-60 cells. Collectively, these results provide evidence that SLURP1 suppresses neutrophil (i) docking on HUVEC cells by decreasing endothelial cell adhesion molecule E-Selectin production, (ii) transmigration through HUVEC monolayer by stabilizing endothelial cell membrane localization of VE-cadherin and β-catenin complex and promoting their barrier function, and (iii) chemotaxis by modulating their polarization and TNF-α-stimulated MMP9 production.

Figure 1

SLURP1 suppresses the interaction of TNF-α-activated dHL-60 and primary human neutrophils with TNF-α-activated HUVEC monolayer. The number of unstimulated or TNF-α-activated (A) dHL-60 cells and (B) primary human neutrophils bound to a confluent HUVEC monolayer upon treatment with control protein (CP; mock purified control protein from the parental strain without SLURP1 expression vector) or SLURP1 is shown. An unpaired t test was used to compare means of CP + TNF-α and SLURP1 + TNF-α and p value is shown in the graph. The data presented is representative of three independent experiments, each with a minimum of four replicates.

Figure 2

SLURP1 suppresses the expression of E-Selectin on HUVEC surface. (A) Comparison of E-Selectin transcript levels in TNF-α-activated HUVEC treated with control protein (CP; mock purified control protein from the parental strain without SLURP1 expression vector) or SLURP1 by QPCR. The data presented is an average of two experiments, each with three replicates. (B–D) Flow cytometry showing percentage of E-Selectin-positive cells and their median fluorescence intensity. In each experiment, 30,000 cells were analyzed. The data presented is representative of three independent experiments with at least two replicates in each. An unpaired t test was used to compare the values obtained with CP + TNF-α and SLURP1 + TNF-α treated HUVEC.

Figure 3

SLURP1 suppresses transmigration of TNF-α-activated dHL-60 through TNF-α-activated HUVEC monolayer, and neutrophil chemotaxis. (A) Number of TNF-α-activated dHL-60 cells transmigrated towards fMLP through a TNF-α-stimulated confluent HUVEC monolayer. The data shown is an average of four independent experiments, each with three replicates. (B,C) The number of (B) dHL-60 cells and (C) primary neutrophils migrated towards fMLP under different conditions tested is shown, quantified using a standard curve. The data shown is an average of three independent experiments, each with three replicates. (D) Phalloidin staining to visualize actin polymerization in dHL60 cells exposed to different conditions. Polarized cells are marked by arrows and those that are not polarized are indicated by arrowheads. (E) Percent of polarized dHL-60 cells under different conditions tested was manually counted. The data presented is the average of three independent experiments, with the polarized cells counted in three different microscopic fields in each experiment. CP, control protein mock purified from the parental strain without SLURP1 expression vector; fMLP, formyl Met-Leu-Phe tripeptide chemoattractant.

Figure 4

SLURP1 promotes VE-Cadherin presence in HUVEC cell junction. Immunofluorescent stain reveals abundant presence of VE-Cadherin (red; indicated by arrowheads) at junctions of confluent HUVEC treated with control protein (CP, control protein mock purified from the parental strain without SLURP1 expression vector) (A), or SLURP1 (B). Activation by TNF-α disrupted VE-Cadherin at HUVEC junctions (open arrows; C), which was restored by treatment with SLURP1 (arrowheads; D). Nuclei are counterstained with DAPI (blue). The data presented is representative of three independent experiments.

Figure 5

SLURP1 suppresses nuclear localization of β-catenin in TNF-α-activated confluent HUVEC monolayer. (A) Immunofluorescent stain with anti-pan β-catenin (upper panels; red), or anti-nuclear β-catenin antibody (lower panels; green) is shown. (B) Fraction of HUVEC with nuclear localization of β-catenin under different conditions tested. Cells with nuclear β-catenin counted by using a program in Metamorph. The data presented is representative of three independent experiments. CP, control protein mock purified from the parental strain lacking SLURP1 expression vector.

Figure 6

SLURP1 suppresses MMP9 expression and activity. (A) QPCR evaluation of MMP9 transcript levels in TNF-α-activated dHL-60 cells in the presence or absence of SLURP1. (B) Representative zymogram showing MMP9 activity in cell culture supernatants under different conditions tested. Relative MMP9 activity under these conditions, quantified by densitometry is shown on the right. The data presented is an average of three independent experiments, each with a minimum of two replicates. CP, control protein mock purified from the parental strain lacking SLURP1 expression vector.

Figure 7

SLURP1 suppresses fMLP-induced AKT phosphorylation. (A) Lysate from dHL-60 cells preincubated with control protein (CP, mock purified control protein from the parental strain lacking SLURP1 expression vector) or SLURP1 and exposed to 1 μM fMLP (formyl Met-Leu-Phe tripeptide chemoattractant) for 2 to 20 minutes was probed with anti-phospho-AKT (S-473) & anti-total AKT antibodies. Full length, uncropped blots can be seen in Supplemental Fig. 3. (B) The ratio of phospho-AKT: total-AKT quantified by densitometry is shown. The data presented is an average of three independent experiments. An unpaired t test was used to compare phospho-AKT: total-AKT ratios in CP- and SLURP1-treated dHL-60 cells at each time point.

Figure 8

Schematic summary of the effects of SLURP1 on neutrophil-endothelial interaction. Data presented in this report demonstrate that SLURP1 acts as an immunomodulatory protein by stabilizing the endothelial barrier function (green arrows), and by suppressing neutrophil (i) binding to endothelial cell surface, (ii) transmigration, (iii) polarization and (iv) chemotaxis (red blunt-ended lines).