Nitric oxide regulates human eosinophil adhesion mechanisms in vitro by changing integrin expression and activity on the eosinophil cell surface

Abstract

1. The nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), inhibits both rat and human eosinophil chemotaxis in vitro. Here, the role of nitric oxide (NO) in human eosinophil cell surface integrin expression and function was investigated. 2. Human peripheral blood eosinophils were treated with L-NAME (0.01 - 1.0 mM) and their adhesion to human fibronectin and serum observed. Adhesion of cells to fibronectin and serum increased by 24.0+/-4.6 and 43.8+/-4.7%, respectively, when eosinophils were treated with 1.0 mM L-NAME. Increased adhesion by L-NAME could be abolished when cells were co-incubated with VLA-4- and Mac-1-specific monoclonal antibodies (mAbs). 3. The NO donor, sodium nitroprusside (2.5 mM), significantly inhibited eosinophil adhesion to fibronectin and serum by 34.3+/-4.5 and 45.2+/-5.6%, respectively. This inhibition was accompanied by a 4 fold increase in the levels of intracellular cyclic GMP. 4. Flow cytometrical analysis demonstrated that L-NAME induced an increased expression of CD11b (Mac-1) on the eosinophil cell surface of 36.3+/-7.4%. L-NAME had no effect upon CD49d (VLA-4) expression. 5. Treatment of human eosinophils, in vitro, with H-[1,2,4] oxadiazolo quinoxalin-1-one (ODQ) (0.1 mM), an inhibitor of soluble guanylate cyclase, also significantly increased eosinophil adhesion to fibronectin and serum by 73.5+/-17.9 and 91.7+/-12.9%, respectively. This increase in adhesion could also be inhibited by co-incubation with the Mac-1 and VLA-4-specific mAbs. 6. In conclusion, results indicate that NO, via a cyclic GMP-dependent mechanism, inhibits the adhesion of human eosinophils to the extracellular matrix (ECM). This inhibition is accompanied by a decrease in the expression and function of the eosinophil's adhesion molecules, in particular, the expression of the Mac-1 integrin and the function of the VLA-4 integrin.

Figure 1

Time course of eosinophil adhesion to fibronectin (20 μg ml⁻¹) and serum (10%, v v^-1)-coated 96 well plates. 3.5×10⁴ cells were added to wells and incubated at 37°C, 5% CO₂. Following incubation, non-adhered cells were removed and percentage cell adhesion was calculated by comparing eosinophil peroxidase activity of adherent cells to that of a standard curve. Results are expressed as mean adhered cell percentages of total cell number±s.e. of three independent experiments with three replicates in each. **P<0.01, ***P<0.001 compared to adhesion at 5 min.

Figure 2

Effect of SNP upon intracellular cyclic GMP levels in eosinophils. Eosinophils (1×10⁷ cells ml⁻¹) were incubated with IBMX (2 mM) for 30 min before treating with SNP for 12 min (37°C) and measuring the concentration of cyclic GMP in 3×10⁶ eosinophils. Results are expressed as mean concentration of cyclic GMP (pmol ml⁻¹) in 3×10⁶ eosinophils±s.e. of three independent experiments with three replicates in each. ***P<0.001 compared to control sample.

Figure 3

Effects of the anti-VLA-4 (HP2/1, 10 μg ml⁻¹), anti-Mac-1 (ICRF 44, 1 : 12 diln) monoclonal antibodies and a non-specific control monoclonal antibody (mAb, 10 μg ml⁻¹) upon human eosinophil adhesion after treatment with L-NAME, 1.0 mM, (a) to 20 μg ml⁻¹ fibronectin, (b) to 10% (v v⁻¹) human serum. Eosinophils (>95% pure) were incubated with monoclonal antibodies (+L-NAME) (25 min, 37°C, 5% CO₂) before allowing cells to adhere to fibronectin/serum-coated plates (15 min, 37°C, 5% CO₂). Results are expressed as mean adhered cell percentages of total cell number±s.e. of four independent experiments with three replicates in each. +P<0.05 compared to untreated cells, **P<0.01, ***P<0.001 compared to cells treated with L-NAME alone (1.0 mM).

Figure 4

Effects of the anti-VLA-4 (HP2/1, 10 μg ml⁻¹), anti-Mac-1 (ICRF 44, 1 : 12 diln) monoclonal antibodies and a non-specific control monoclonal antibody (mAb, 10 μg ml⁻¹), upon ODQ-treated human eosinophil adhesion to (a) 20 μg ml⁻¹ fibronectin, (b) 10% (v v⁻¹) serum. Eosinophils (>95% pure) were incubated with ODQ/monoclonal antibodies (25 min, 37°C, 5% CO₂) before allowing cells to adhere to ligand-coated plates (15 min, 37°C, 5% CO₂). Results are expressed as mean adhered cell percentages of total cell number±s.e. of three independent experiments with three replicates in each ++P<0.01, +++P<0.001 compared to untreated cells, **P<0.01, ***P<0.001 compared to cells treated with ODQ alone.