A novel mechanism of neutrophil recruitment in a coculture model of the rheumatoid synovium

Abstract

Objective: Rheumatoid arthritis (RA) is classically thought of as a Th1, T lymphocyte-driven disease of the adaptive immune system. However, cells of the innate immune system, including neutrophils, are prevalent within the diseased joint, and accumulate in large numbers. This study was undertaken to determine whether cells of the rheumatoid stromal microenvironment could establish an inflammatory environment in which endothelial cells are conditioned in a disease-specific manner to support neutrophil recruitment.

Methods: Human umbilical vein endothelial cells (ECs) and fibroblasts isolated from the synovium or skin of RA patients were established in coculture on opposite sides of porous transwell filters. After 24 hours of EC conditioning, the membranes were incorporated into a parallel-plate, flow-based adhesion assay and levels of neutrophil adhesion to ECs were measured.

Results: ECs cocultured with synovial, but not skin, fibroblasts could recruit neutrophils in a manner that was dependent on the number of fibroblasts. Antibody blockade of P-selectin or E-selectin reduced neutrophil adhesion, and an antibody against CD18 (the beta2 integrin) abolished adhesion. Blockade of CXCR2, but not CXCR1, also greatly inhibited neutrophil recruitment. Interleukin-6 (IL-6) was detectable in coculture supernatants, and both IL-6 and neutrophil adhesion were reduced in a dose-dependent manner by hydrocortisone added to cocultures. Antibody blockade of IL-6 also effectively abolished neutrophil adhesion.

Conclusion: Synovial fibroblasts from the rheumatoid joint play an important role in regulating the recruitment of inflammatory leukocytes during active disease. This process may depend on a previously unsuspected route of IL-6-mediated crosstalk between fibroblasts and endothelial cells.

Figure 1

Confirmation of fibroblast phenotype. Fibroblasts from 4 patients with rheumatoid arthritis (RA) were isolated from synovium (RASY) or skin (RADF) obtained at total knee arthroplasty. Cells were stained for the fibroblast marker fibronectin.

Figure 2

Assembly of coculture inserts. A, A porous polyethylene terepthalate culture insert on which cocultures were established by growing fibroblast and endothelial cells (ECs) on opposite sides of the porous membrane. B, The parallel-plate, flow-based adhesion assay assembled with coculture in situ. C, The components of the assay included an upper Perspex plate (i) that had a machined recess to accept a glass coverslip (ii), which formed the upper surface of the flow channel. A silicone gasket (iii) defined the depth of the flow channel (150 μm). The lower acetal plastic plate (iv) formed the lower surface of the flow channel and had a recess into which the culture insert fit so that the ECs also formed part of the lower surface of the flow channel. The insert (v) was held in place by a Perspex plate (vi) and a smaller silicone gasket (vii). Inlet and outlet conduits (viii) allowed the flow of leukocyte suspension or wash buffer through the flow channel. The plates were held together firmly by locating screws (ix and x).

Figure 3

Adhesion of flowing neutrophils to endothelial cells (ECs) cocultured with fibroblasts. A, A comparison of synovial (RASY) and skin (RADF) fibroblast explants from 5 different patients with rheumatoid arthritis (RA). B, The mean ± SEM response of ECs cultured with fibroblasts from the 5 donors. C, Effects of varying the number of fibroblasts in coculture with ECs. Values are the mean ± SEM in the 5 donors.

Figure 4

Levels of neutrophil adhesion achieved with antibody blockade. A, Effects of anti–E-selectin, anti–P-selectin, a combination of these antibodies, a control antibody, or blockade of CD18 (β2 integrin). B, Neutrophil adhesion levels with the use of anti-CXCR1, anti-CXCR2, or a combination of these antibodies. C, Effects of anti–epithelial neutrophil–activating peptide 78 (ENA-78), growth-related oncogene α (GROα), or interleukin-8 (IL-8) (all CXC chemokines). Values are the mean and SEM from at least 4 experiments using different endothelial cells and fibroblast explants. RASY = fibroblasts isolated from synovium of rheumatoid arthritis patients. * = P < 0.05 versus untreated control, by Student’s paired t-test.

Figure 5

Role of hydrocortisone and interleukin-6 (IL-6) activity in neutrophil adhesion. A and B, Effects of hydrocortisone on A, the adhesion of neutrophils to ECs cocultured with RASY and RADF, and B, the levels of IL-6 released into the medium by cocultured cells. Values are the mean and SEM from at least 4 experiments using different ECs and fibroblast explants. C, Correlation between levels of IL-6 released into coculture medium and levels of neutrophil adhesion to ECs cocultured with fibroblasts. See Figure 3 for other definitions.

Figure 6

Effect of antibody blockade of the biologic function of interleukin-6 (IL-6), tumor necrosis factor α (TNFα), or IL-1β on neutrophil adhesion. Values are the mean and SEM levels of neutrophil adhesion. * = P < 0.05, by Pearson’s correlation test assuming a Gaussian distribution.