Characterization of autoantibodies to endothelial cells in systemic sclerosis (SSc): association with pulmonary fibrosis

Abstract

To determine the prevalence and the characterization of antibodies to endothelial cells in patients with SSc, serum samples from 80 patients with SSc, 20 patients with systemic lupus erythematosus (SLE), and 20 healthy control subjects were examined by ELISA using cultured human umbilical vein endothelial cells (HUVEC), indirect immunofluorescence analysis (IIF), and immunoblotting using cytoplasmic extract of HUVEC. IgG and/or IgM isotype anti-endothelial cell antibodies (AECA) were demonstrated by ELISA in 43 of 80 patients with SSc (54%), in 15 of 20 patients with SLE (75%), and in none of 20 healthy control subjects. Immunofluorescence analysis on HUVEC substrate showed homogeneous cytoplasmic staining. Immunoblotting demonstrated that these patients had antibodies directed to one or several antigens of approximately 60, 90, 110 and 140 kD, and the most common responses were to the 90-kD antigen. By the immunofluorescence method using HUVEC, affinity-purified anti-90-kD antibodies showed identical cytoplasmic staining to that produced by sera positive for AECA. Furthermore, AECA were closely correlated with pulmonary fibrosis in patients with SSc. These findings suggest that patients with SSc have abnormal antibodies to endothelial cell antigens, and support the hypothesis that endothelial dysfunction is involved in the development of this disease.

Fig. 1

Levels of anti-endothelial cell antibodies (AECA) in serum samples of patients with SSc, those with systemic lupus erythematosus (SLE) and healthy control subjects (Normal). Optical density (OD) values determined in an ELISA are shown on the ordinate. The horizontal lines mark the cut-off levels of normal serum binding.

Fig. 2

Indirect immunofluorescence localization of antigens targeted by anti-endothelial cell antibodies (AECA) in cultured human umbilical vein endothelial cells (HUVEC). Cytoplasmic homogeneous staining was detected in HUVEC (a), while this staining was not detected in cultured human dermal fibroblasts (b), or in cultured human keratinocytes (c). No staining was shown using control sera (d) or secondary antibody (e). Affinity-purified anti-90-kD antibodies reacted with determinants localized to the cytoplasm of HUVEC (f).

Fig. 3

Representative results of immunoblotting. Sera from patients positive for anti-endothelial cell antibodies (AECA) predominantly reacted with one or more antigens of approximately 60, 90, 110, and 140 kD (lanes 1–6), but not with cytoplasmic antigens of human dermal fibroblasts (lane 9). Serum samples from control subjects did not react with cytoplasmic antigens of HUVEC (lane 7), nor with those of human dermal fibroblasts (lane 10). Lane 8, colloidal gold staining of cytoplasmic antigens of HUVEC.