The Phenotype and Secretory Activity of Adipose-Derived Mesenchymal Stem Cells (ASCs) of Patients with Rheumatic Diseases

Abstract

Mesenchymal stem/stromal cells (MSCs) have immunosuppressive and regenerative properties. Adipose tissue is an alternative source of MSCs, named adipose-derived mesenchymal stem cells (ASCs). Because the biology of ASCs in rheumatic diseases (RD) is poorly understood, we performed a basic characterization of RD/ASCs. The phenotype and expression of adhesion molecules (intracellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1) on commercially available healthy donors (HD), ASC lines (n = 5) and on ASCs isolated from patients with systemic lupus erythematosus (SLE, n = 16), systemic sclerosis (SSc, n = 17) and ankylosing spondylitis (AS, n = 16) were analyzed by flow cytometry. The secretion of immunomodulatory factors by untreated and cytokine-treated ASCs was measured by ELISA. RD/ASCs have reduced basal levels of CD90 and ICAM-1 expression, correlated with interleukin (IL)-6 and transforming growth factor (TGF)-β1 release, respectively. Compared with HD/ASCs, untreated and tumour necrosis factor (TNF) + interferon (IFN)-γ (TI)-treated RD/ASCs produced similar amounts of prostaglandin E2 (PGE₂), IL-6, leukemia inhibiting factor (LIF), and TGF-β1, more IL-1Ra, soluble human leukocyte antigen G (sHLA-G) and tumor necrosis factor-inducible gene (TSG)-6, but less kynurenines and galectin-3. Basal secretion of galectin-3 was inversely correlated with the patient's erythrocyte sedimentation rate (ESR) value. IFN-α and IL-23 slightly raised galectin-3 release from SLE/ASCs and AS/ASCs, respectively. TGF-β1 up-regulated PGE₂ secretion by SSc/ASCs. In conclusion, RD/ASCs are characterized by low basal levels of CD90 and ICAM-1 expression, upregulated secretion of IL-1Ra, TSG-6 and sHLA-G, but impaired release of kynurenines and galectin-3. These abnormalities may modify biological activities of RD/ASCs.

Keywords: adipose-derived mesenchymal stem cells; ankylosing spondylitis; phenotype; secretory potential; systemic lupus erythematosus; systemic sclerosis.

Figure 1

The phenotype of ASCs from healthy donors (HD) and patients with rheumatic diseases (RD). Expression of MSC specific (A,B) and non-specific (C,D) markers was assessed quantitatively (B) and/or qualitatively (A,C,D) using ASCs from healthy donors (HD/ASCs; n = 5), systemic lupus erythematosus (SLE/ASCs; n = 14), systemic sclerosis (SSc/ASCs; n = 13) and ankylosing spondylitis (AS/ASCs; n = 12) patients. Data are expressed as the median (horizontal line) with interquartile range (IQR, box), lower and upper whiskers (data within 3/2xIQR) and outliers (points) (Tukey’s box) (A,B) or as the median with IQR (C,D). ^# p = 0.05–0.01; ^## p = 0.01–0.001 for HD/ASCs versus RD/ASCs comparison. Pearson’s (R) and Spearman’s rank (R_s) correlation coefficients. Solid cirle—HD/ASC samples, hollow circles RD/ASC samples (E,F).

Figure 2

Expression of adhesion molecules on untreated and cytokine-treated ASCs. Healthy donors (HD; n = 5), systemic lupus erythematosus (SLE; n = 16), systemic sclerosis (SSc; n = 16) and ankylosing spondylitis (AS; n = 15) ASCs were cultured in medium alone (untreated ASCs) or in the presence of tumor necrosis factor (TNF)-α and interferon (IFN)-γ (TI treated ASCs) for 24 h. The qualitative (A,B) and quantitative (C,D) assessments of intracellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 expression were performed. Results are expressed as the Tukey’s boxes (see Figure 1). ^# p = 0.05–0.01, ^## p = 0.01–0.001 for HD/ASCs versus patients’ ASCs, while * p = 0.05–0.01, ** p = 0.01–0.001, *** p = 0.001–0.0001 for untreated versus TI treated ASC comparisons, + p = 0.05–0.01 for patients’ versus patients’ ASCs. Solid circles (points) represents outliers.

Figure 3

Impaired secretion of galectin-3 and kynurenines by ASCs of patients with rheumatic diseases (RD). Five healthy donor (HD) ASCs lines from two different passages (n = 10), systemic lupus erythematosus (SLE; n = 16), systemic sclerosis (SSc; n = 16–17) and ankylosing spondylitis (AS; n = 15) patients’ ASCs were cultured in medium alone (C, control), or in the presence of the indicated cytokines (TI, TNF-α + IFN-γ). Galectin-3 and kynurenine concentrations were measured in culture supernatants. (A,B) Data are expressed as Tukey’s boxes (see Figure 1); * p = 0.05–0.01, *** p = 0.001–0.0001 for control versus cytokine-treated cells; ^# p = 0.05–0.01, ^## p = 0.01–0.001, ^### p = 0.001–0.0001, ^#### p < 0.0001 for HD/ASCs versus RD/ASCs comparisons. (C,D) R_s, Spearman’s rank correlation coefficient. Hollow circles represents RD/ASC samples, solid circle represents SSc/ASC samples. ESR - erythrocyte sedimentation rate.

Figure 4

Enhanced secretion of IL-1Ra, soluble human leukocyte antigen G (sHLA-G) and tumor necrosis factor-inducible gene (TSG)-6 by ASCs of patients with rheumatic diseases (RD). Culture conditions of ASC lines from healthy donors (HD; n = 10), systemic lupus erythematosus (SLE; n = 12–16), systemic sclerosis (SSc; n = 10–17) and ankylosing spondylitis (AS; n = 9–15) patients were the same as described in Figure 3. Concentrations of indicated factors were measured in culture supernatants. Data are expressed as the Tukey’s boxes (see Figure 1). No differences between untreated (C) and cytokine-stimulated cells were found. ^# p = 0.05–0.01; ^## p = 0.01–0.001; ^### p = 0.001–0.0001; ^#### p < 0.0001 for HD/ASCs versus RD/ASCs comparison.

Figure 5

Similar secretion of other factors by ASCs of healthy donors and patients with rheumatic diseases. Culture conditions of ASC lines from healthy donors (HD; n = 10) and from patients with rheumatic diseases (RD), i.e., systemic lupus erythematosus (SLE; n = 16), systemic sclerosis (SSc; n = 16–17) and ankylosing spondylitis (AS; n = 15), were the same as described in Figure 3. Concentrations of indicated factors were measured in culture supernatants. Data are expressed as the Tukey’s boxes (see Figure 1). * p = 0.05–0.01, *** p = 0.001–0.0001 for untreated (C) versus cytokine-treated cells; ^# p = 0.05–0.01, ^## p = 0.01–0.001 for HD/ASCs versus RD/ASCs.