The human glomerular endothelial cells are potent pro-inflammatory contributors in an in vitro model of lupus nephritis

Abstract

Juvenile-onset lupus nephritis (LN) affects up to 80% of juvenile-onset systemic lupus erythematosus patients (JSLE). As the exact role of human renal glomerular endothelial cells (GEnCs) in LN has not been fully elucidated, the aim of this study was to investigate their involvement in LN. Conditionally immortalised human GEnCs (ciGEnCs) were treated with pro-inflammatory cytokines known to be involved in LN pathogenesis and also with LPS. Secretion and surface expression of pro-inflammatory proteins was quantified via ELISA and flow cytometry. NF-κΒ and STAT-1 activation was investigated via immunofluorescence. Serum samples from JSLE patients and from healthy controls were used to treat ciGEnCs to determine via qRT-PCR potential changes in the mRNA levels of pro-inflammatory genes. Our results identified TNF-α, IL-1β, IL-13, IFN-γ and LPS as robust in vitro stimuli of ciGEnCs. Each of them led to significantly increased production of different pro-inflammatory proteins, including; IL-6, IL-10, MCP-1, sVCAM-1, MIP-1α, IP-10, GM-CSF, M-CSF, TNF-α, IFN-γ, VCAM-1, ICAM-1, PD-L1 and ICOS-L. TNF-α and IL-1β were shown to activate NF-κB, whilst IFN-γ activated STAT-1. JSLE patient serum promoted IL-6 and IL-1β mRNA expression. In conclusion, our in vitro model provides evidence that human GEnCs play a pivotal role in LN-associated inflammatory process.

Figure 1

LN urinary biomarker, chemokine, cytokine and growth factor secretion after 24-hour ciGEnC-stimulation with individual cytokines, combination of all cytokines (All) and LPS. MCP-1 (a), sVCAM-1 (b), IL-6 (c), IL-8 (d), IL-10 (e), M-CSF (f), GM-CSF (g), MIP-1α (h), IP-10 (i), TNF-α (j) and IFN-γ (k) secretion in response to cytokine treatments. MCP-1 (l) and M-CSF secretion (m) following combined TNF-α and IL-1β treatments. N = 5–6/group, Data are presented as median concentrations (pg/ml) [range] are analysed using Kruskal-Wallis test with Dunn’s post-hoc test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs untreated.

Figure 2

Effect of 24 h ciGEnC treatments with 5% JSLE or HC sera in pro-inflammatory gene mRNA expression. Changes in IL-6 (a), IL-8 (b), IL-10 (c), M-CSF (d), GM-CSF (e), MCP-1 (f), VCAM-1 (g), MIP-1α (h), IP-10 (i), TNF-α (j), IL-1β (k) and NF-κB (l) mRNA expression levels. Data presented as median ΔΔCt [range] are analysed with Mann-Whitney test. *P </= 0.05 vs HC N = 10–20/group.

Figure 3

Effect of 24 h ciGEnC treatments with 5% active and inactive LN or HC sera in pro-inflammatory gene mRNA expression. Changes in IL-6 (a), IL-8 (b), IL-10 (c) M-CSF (d), GM-CSF (e), MCP-1 (f), VCAM-1 (g), MIP-1α (h), IP-10 (i), TNF-α (j), IL-1β (k) and NF-κB (l) mRNA expression levels. Data presented as median ΔΔCt [range] are analysed with Kruskal-Wallis with Dunn’s post-hoc test. *P </= 0.05. N = 10/group.

Figure 4

Effect of 24 h ciGEnC treatments with 5% active and inactive LN or HC sera in IL-1β and IL-6 secretion. IL-1β levels in active and inactive LN patient and HC sera (a) and IL-1β levels in ciGEnC conditioned media (b). IL-6 levels in active and inactive LN patient and HC sera (c) and IL-6 levels in ciGEnC conditioned media (d). Data presented as median concentrations (pg/ml) [range] are analysed using Kruskal-Wallis test with Dunn’s post-hoc test. *P </= 0.05, **P < 0.01 vs HC or vs untreated ciGEnC conditioned media. N = 6/group.

Figure 5

VCAM-1, ICAM-1, PD-L1 and ICOS-L surface expression following 24-hour stimulation with cytokines and LPS. (a) 24-hour VCAM-1 surface expression following IL-13 and TNF-α treatments. (b) 24-hour VCAM-1 surface expression following IL-1β, IFN-γ and LPS treatments. (c) 24-hour ICAM-1 surface expression following IL-13 and TNF-α treatments. (d) 24-hour ICAM-1 surface expression IL-1β, IFN-γ and LPS treatments. (e) 24-hour PD-L1 surface expression following TNF-α, IL-1β, IL-13, IFN-γ and LPS treatments. (f) 24-hour ICOS-L surface expression following TNF-α, IL-1β, IL-13, IFN-γ and LPS treatments. N = 4–6/group. Data presented as median [range] are analysed using Kruskal-Wallis test with Dunn’s post-hoc test. *P </= 0.05, **P < 0.01, ***P < 0.001. N = 4–6/group.

Figure 6

Neutrophil adhesion assay following 24 h stimulation of ciGEnCs with TNF-α and IL-13. (a) Neutrophil adhesion on untreated ciGEnCs. (b) Neutrophil adhesion on TNF-α-treated ciGEnCs. (c) Neutrophil adhesion on IL-13-treated ciGEnCs. (d) Neutrophil adhesion on TNF-α + IL-13-treated ciGEnCs. White arrow on (a) indicates neutrophil. (e) Graph of statistical analysis of neutrophil adhesion assay. Data presented as median cell number [range] are analysed using Kruskal-Wallis test with Dunn’s post-hoc test. *P </= 0.05 vs untreated. N = 6/group. Scale bars: 200 μm.

Figure 7

Western blotting for Iκ-Bα protein expression, 20x immunofluorescence images of ciGEnCs and diagrams of statistical analysis for NF-κB, STAT-1 and STAT-2 activation and nuclear translocation, after 30 minutes of stimulation with cytokines and LPS. (a) Representative image and graph of Iκ-Bα and beta-actin Western blots (blots have been cropped), following 30-minute stimulation. Data presented as (Mean +/− SEM) in ratio diagrams are analysed using Friedman test with Dunn’s post-hoc test, *P < 0.05 vs untreated. (b) Treatments of ciGEnCs with TNF-α, IL-1β and LPS for 30 minutes for NF-κB nuclear translocation. (c) Treatments of ciGEnCs with IFN-γ for 30 minutes for STAT-1 nuclear translocation. (d) Treatments of ciGEnCs with IFN-γ for 30 minutes for STAT-2 nuclear translocation. Data (r-values for nuclear co-localisation of DAPI and A488 or A568 for each treatment group) representative of three similar experimental repeats are presented as box and whisker plots and are analysed using Kruskal-Wallis test with Dunn’s post-hoc test. ****P < 0.0001 vs untreated. Scale bars: 100 μm.

Figure 8

Potential effect of stimulated GEnCs in LN based on the in vitro findings. Under the highly inflammatory environment created within the glomeruli by the presence of TNF-α, IL-1β, IL-13 and IFN-γ but also by the potential presence of LPS, the human GEnCs will be activated to produce, secrete and express a variety of pro-inflammatory cytokines, chemokines, blood cell growth factors and adhesion molecules that will further exacerbate the renal disease by either promoting the infiltration of immune cells within the glomeruli or by activating their neighbouring renal cells, the podocytes and mesangial cells.