Tumour necrosis factor alpha stimulated rheumatoid synovial microvascular endothelial cells exhibit increased shear rate dependent leucocyte adhesion in vitro

Abstract

Objective: To investigate endothelial cell adhesion molecule expression and leucocyte adhesion to endothelial cells isolated from the microvasculature of rheumatoid arthritic synovial tissue (SMEC) in comparison with similar cells isolated from healthy subcutaneous adipose tissue (ADMEC) or from umbilical veins (HUVEC).

Methods: Cultured endothelial cells were treated with tumour necrosis factor alpha (TNFalpha) for 2-24 hours before the assessment of cell surface E-selectin, vascular (VCAM-1) or intercellular cell adhesion molecule-I (ICAM-1) expression. Neutrophil and T lymphocyte adhesion to TNFalpha treated endothelial cells was assessed using static and shear dependent assay systems.

Results: VCAM-1 expression by SMEC was significantly less sensitive to TNFalpha stimulation than HUVEC or ADMEC. E-selectin expression by SMEC appeared to be more sensitive to TNFalpha stimulation and maximal expression was about 30% greater in comparison with HUVEC or ADMEC. Sensitivity to TNFalpha induction and maximal ICAM-1 expression was similar in all three endothelial cell types. Static neutrophil adhesion to TNFalpha stimulated SMEC was significantly increased in comparison with HUVEC, however this phenomenon was dependent on the presence of neutralising antibodies to ICAM-1. At shear rates in excess of 2.4 dynes/cm(2) significantly more neutrophils and, predominantly CD45RO+, T lymphocytes adhered to TNFalpha stimulated SMEC than HUVEC.

Conclusion: Rheumatoid synovial endothelial cells differentially regulate E-selectin and VCAM-1. The increased ability of TNFalpha stimulated synovial endothelial cells to support leucocyte adhesion may help to explain the leucocyte, in particular CD45RO+ T-lymphocyte, recruitment observed in the rheumatoid synovium.

Figure 1

Schematic representation of the parallel plate flow cell used. The convergence of the side walls of the cell increases the velocity of the perfusate as it travels through the cell. This increase in velocity permits measurements to be made on cells exposed to an increasing wall shear stress in the same system without changing the mass flow volume. Wall shear stress at position A (τ_A) = 2.4 dynes/cm² and τ_B = 4.0 dynes/cm² assuming a volumetric flow rate of 0.916 cm^-3/sec (55 ml/min).

Figure 2

(A) HUVEC, ADMEC and SMEC monolayers were stimulated TNFα (0-100 IU/ml) for six hours before assessment of E-selectin expression. Results are normalised as percentages of the optical density observed in SMEC cultures stimulated with 100 IU/ml TNFα and depict 95% confidence intervals for the mean response. E-selectin expression by SMEC was compared with both HUVEC and ADMEC. SMEC display significantly (p<0.05*, p<0.01**, p<0.001***) higher E-selectin expression than HUVEC or ADMEC at all concentrations of TNFα, (n=3). (B) HUVEC, ADMEC and SMEC monolayers were pre-incubated with TNFα (0-100 IU/ml) for six hours before assessment of VCAM-1 expression. Results are normalised as percentages of the optical density observed in SMEC cultures stimulated with 100 IU/ml TNFα and depict 95% confidence intervals for the mean response. VCAM-1 expression by HUVEC and ADMEC was not significantly different. SMEC display significantly (p<0.001***) lower VCAM-1 expression than HUVEC or ADMEC at concentrations of TNFα in excess of 1 IU/ml, (n=3).

Figure 3

(A) Neutrophil adhesion to HUVEC and SMEC monolayers pre-incubated with TNFα (0-100 IU/ml). Results show mean binding of neutrophils as a percentage of the initial number added and depict 95% confidence intervals for the mean binding. SMEC occasionally, but not consistently, bind significantly (p<0.01**) higher numbers of neutrophils than HUVEC (n=5). (B) T lymphocyte adhesion to HUVEC and SMEC monolayers pre-incubated with TNFα (0-100 IU/ml). Results are expressed as the mean percentage binding of the original number of lymphocytes added and depict 95% confidence intervals for the mean binding. No significant differences were observed between cell types (n=5). (C) Comparison of static PMN adhesion to TNFα (0-100 IU/ml) stimulated endothelial cell monolayers. Results show mean binding of neutrophils as a percentage of the initial number added and depict 95% confidence intervals for the mean binding. Results indicate differences in neutrophil binding to the two endothelial cell types before the addition of anti-ICAM-1 antibody (50 µg/ml), rarely reaches significance (p<0.05*). However, in the presence of anti-ICAM-1 antibody SMEC are capable of binding significantly (p<0.05*, p<0.01**, p<0.001***) more PMN than similarly treated HUVEC at a number of TNFα concentrations (n=3).

Figure 4

Adhesion of (A) peripheral blood neutrophils and (B) T lymphocytes to SMEC and HUVEC monolayers pre-stimulated with TNFα (50 IU/ml) for five hours. The cell counts represents the mean number of leucocytes (per mm²) attached to the endothelial monolayers, after 30 minutes of continuous circulation, at points in the flow chamber where wall shear rates were calculated at 2.4 and 4.0 dynes/cm² respectively and depict 95% confidence intervals for the mean response. SMEC monolayers bound significantly (p<0.05*) higher numbers of both neutrophils at both 2.4 and 4.0 dynes/cm² in comparison with HUVEC at the same rates of shear. Differences in T lymphocytes adhesion to SMEC in comparison with HUVEC were only significant (p<0.05*) at higher (4.0 dynes/cm²) rates of shear (n=3). (C) Immunohistochemical analysis of adherent T lymphocyte phenotype and initial proportions in healthy peripheral blood. Results are expressed as the proportion of cells bearing a CD45RO+ phenotype as a percentage of the total and depict 95% confidence intervals for the mean response. Cell populations adherent to both SMEC and HUVEC monolayers at both 2.4 and 4.0 dynes/cm² display a significant (p<0.01**) increase in the proportions of CD45RO+ cells in comparison with peripheral blood. SMEC also display a significantly (p<0.05*) increased capacity to bind such cells in comparison to HUVEC at higher (4.0 dynes/cm²) rates of shear (n=3).