Increased transendothelial migration of scleroderma lymphocytes

Abstract

Background: CD4+ T lymphocytes play an important part in the pathogenesis of scleroderma (systemic sclerosis, SSc) and predominate in perivascular SSc skin lesions. Both soluble and membrane bound adhesion molecules are overexpressed in SSc, possibly influencing lymphocyte/endothelial cell (EC) contact.

Objective: To assess the transendothelial migration capacity of peripheral lymphocytes in vitro.

Patients and methods: Collagen was covered with human umbilical vein endothelial cells (HUVEC), and peripheral blood mononuclear cells (PBMC) of patients and matched healthy controls (HC) were added in parallel experiments. Before and after fractionated harvest of non-adherent, bound, and migrated lymphocytes, the CD4/CD8 ratio and the lymphocytic expression of activation markers and adhesion molecules were analysed by fluorocytometry.

Results: 13 (SD 12)% of the SSc PBMC migrated compared with only 5 (5)% HC PBMC (p<0.0002); this increase was primarily due to the migration of CD3+ T lymphocytes and mainly to a larger proportion of CD4+ cells within this CD3+ fraction (71 (SD 14)% for SSc v 56 (14)% for HC, p<0.03), leading to an increased CD4/CD8 ratio among migrated SSc lymphocytes in comparison with controls (3.3 (1.5) v 1.62 (0.93), p<0.006). Among migrated SSc CD4+ T lymphocytes, the frequency of HLA-DR+ cells was increased; migrated lymphocytes highly expressed the adhesion molecules CD11a, CD49d, CD29, and CD44.

Conclusion: Transendothelial migration of CD4+ T lymphocytes is enhanced in SSc, and migrating cells exhibit an activated phenotype. The data suggest that activated CD3+CD4+ lymphocytes as found in SSc peripheral blood are prone to transvascular migration, thus contributing to the formation of typical perivascular lymphocytic infiltrates.

Figure 1

Assay of transendothelial migration. HUVEC after the third to fourth passage formed confluent monolayers on collagen gels after overnight incubation. On the second day of the experiment, we added fresh PBMC, obtained from SSc and matched HC and processed them in parallel experiments. After 1 hour of migration we analysed the harvested cell fractions (NAD, BND, MIG) and ex vivo stained PBMC by fluorocytometry. NAD, non-adherent; BND, bound; MIG, migrated, EC, endothelial cells, PBMC peripheral blood mononuclear cells.

Figure 2

Increased transendothelial migration of SSc PBMC. Testing PBMC of SSc and HC in parallel experiments. Filled triangles indicate diffuse SSc, open triangles indicate limited disease, and the small circle indicates that the respective negative control had highly activated lymphocytes and developed influenza the next day. This single sample was therefore excluded from all analyses.

Figure 3

Rise in the CD4/CD8 ratio in migrated SSc lymphocytes. The CD4/CD8 ratio in patients with SSc was only slightly raised ex vivo (p = NS), but increased in 8/12 patients with SSc and decreased in 10/11 HC, leading to a significantly raised CD4/CD8 ratio in migrated lymphocytes of patients with SSc when compared with HC (for details see table 2).