Interleukin 10 hampers endothelial cell differentiation and enhances the effects of interferon α on lupus endothelial cell progenitors

Abstract

Objective: SLE is an autoimmune disease characterized by autoantibody generation, organ damage and an increased risk of cardiovascular disease. Generally considered an anti-inflammatory cytokine, IL-10 is increased in SLE and correlates with poor cardiovascular outcomes in the general population. The aim of this study was to explore the putative role of IL-10 in modulating endothelial function in SLE by examining the effects of this cytokine on endothelial progenitor cell/circulating angiogenic cell (EPC/CAC) differentiation.

Methods: Human and murine control and lupus EPCs/CACs were differentiated into mature endothelial cells (ECs) in the presence or absence of graded concentrations of recombinant IL-10 with or without recombinant IFN-α or a neutralizing antibody to IL-10. IL-10-deficient mice were examined to assess the role of this cytokine in type I IFN-mediated inhibition of EC differentiation and neo-angiogenesis using an in vivo Matrigel plug assay. Serum IL-10 concentrations were measured via ELISA.

Results: IL-10 hampers EC differentiation in a dose-dependent manner. In murine EPC cultures, IL-10 is required to observe the inhibitory effects of type I IFNs on EPC function and neo-angiogenesis. In human SLE EPC/CAC cultures, neutralization of IL-10 significantly improved the differentiation of EPCs, and IL-10 enhanced type I IFN-mediated EPC/CAC dysfunction. The presence of IL-10 in serum inversely correlated with EPC/CAC function in SLE but not in control cells.

Conclusion: IL-10 interferes with endothelial differentiation and may enhance the effects of type I IFN on vascular repair in SLE. IL-10 may be a relevant target for improving cardiovascular risk in SLE.

Keywords: IL-10; cardiovascular; endothelial progenitor; interferon α; lupus.

Fig. 1

IL-10 inhibits human EPC differentiation in a dose-dependent manner Human control EPCs were incubated with 1000 U/ml recombinant IFN-α or graded concentrations of recombinant IL-10 and cultured under pro-angiogenic conditions. After 2 weeks in culture, mature ECs were quantified as those that co-stained with ac-LDL and FITC-UEA lectin using live fluorescent microscopy imaging. (A) Results represent the mean (s.e.m.) of double-positive ECs per high-power field (n = 18). (B) Representative microscopic images displaying double-stained mature ECs. (C) SLE (n = 18) and control (n = 18) EPC/CACs were cultured in the presence of graded concentrations of recombinant IL-10 and results are reported as in Fig. 1A. *P < 0.05, **P < 0.01, ***P < 0.001. ac-LDL: acetylated low-density lipoprotein; CAC: circulating angiogenic cell; EC: endothelial cell; EPC: endothelial progenitor cell; UEA: Ulex europaeus agglutinin 1.

Fig. 2

IL-10 is required for the inhibitory effects of type I IFN on murine EPC differentiation and in vivo neo-angiogenesis (A) Wild-type (WT) murine EPCs were incubated with 1000 U/ml IFN-α in the presence or absence of a neutralizing IL-10 antibody or isotype control. Following 7 days of culture, mature ECs were quantified as in Fig. 1 using BS-1 instead of UEA-lectin. Results represent the mean (s.e.m.) of mature ECs quantified in triplicate from three different mice. (B) WT (n = 4) or IL-10^−/− (n = 6) EPCs were incubated with IFN-α and mature ECs were quantified as in Fig. 2A. (C) Representative images (total magnification 100 × ) of murine EC cultures from Fig. 2B (green: BS-lectin; red: ac-LDL). (D) Human EPC/CAC cultures (n = 6) were treated as in Fig. 2A utilizing a neutralizing antibody specific for human IL-10. On day 14 of culture, mature ECs were quantified as in Fig. 1. **P ≤ 0.01, NS: non-significant. (E and F) Matrigel embedded with 20 nM fibroblast growth factor with or without 1000 U/ml IFN-α was injected subcutaneously into WT (n = 13) or IL-10^−/− (n = 8) mice. Seven days after injection, Matrigel plugs were harvested and their HgB content was quantified as an estimate of neo-angiogenesis within the plug. Results in Fig. 2A represent HgB quantification. Results in Fig. 2B show the percentage inhibition of angiogenesis in plugs containing IFN-α as compared with plugs not exposed to IFN-α. *P < 0.05. BS-1: Bandeiraea (Griffonia) simplicifolia lectin 1; CAC: circulating angiogenic cell; EPC: endothelial progenitor cell; HgB: haemoglobin; UEA: Ulex europaeus agglutinin 1.

Fig. 3

IFN-α + IL-10 is detrimental to SLE but not control EPC differentiation EPC/CACs from SLE (n = 18) and control (n = 18) subjects were incubated with 1000 U/ml IFN-α, 30 ng/ml IL-10 or both and mature EC differentiation was assessed as in Fig. 1. Results are presented as the mean (s.e.m.) percentage inhibition of EPC differentiation compared with autologous untreated cells. *P < 0.05, **P < 0.01. CAC: circulating angiogenic cell; EC: endothelial cell; EPC: endothelial progenitor cell.

Fig. 4

Endogenous IL-10 impairs SLE EPC function and negatively correlates with EPC differentiation in SLE but not control samples (A) Control (n = 5) or SLE (n = 11) EPC/CACs were cultured in the presence of a neutralizing antibody to IL-10 or an isotype control. On day 14, mature EC differentiation was assessed as in Fig. 1A. (B) Representative photomicrographs of day 14 mature EC cultures from Fig. 4A (red: ac-LDL; green: UEA-lectin). (C) Serum concentrations of IL-10 of control (n = 28) and SLE (n = 29) serum were determined by ELISA. Comparisons between serum levels were made via Mann–Whitney U-test. (D) IL-10 as determined in Fig. 2C was correlated with mature EC numbers derived from untreated control and SLE EPCs/CACs from each subject and linear regression was performed. For control samples, P = 0.5597 and Pearson’s r = − 0.1249. For SLE samples, P = 0.0016 and Pearson’s r = −0.5674. Statistical significance was validated via quasi-Poisson regression: for control, P = 0.598; for SLE, P = 0.0015. ac-LDL: acetylated low-density lipoprotein; CAC: circulating angiogenic cell; CNTL: control; EC: endothelial cell; EPC: endothelial progenitor cell; UEA: Ulex europaeus agglutinin 1.

Fig. 5

Summation of the effects of IL-10 in murine and human EPC cultures In mice, IL-10 acts as an intermediary for the inhibitory effects of IFN-α on vasculogenesis and EPC differentiation. In contrast, in human EPC cultures, IL-10 is not required for the effects of IFN-α on EPC function. In control cultures, IL-10 is protective in the presence of IFN-α, but in SLE patients, IL-10 and IFN-α promote EPC/CAC dysfunction and the combination of the cytokines remains detrimental. CAC: circulating angiogenic cell; EPC: endothelial progenitor cell.