Endothelial-derived thrombospondin-1 promotes macrophage recruitment and apoptotic cell clearance

Abstract

Rapid apoptotic cell engulfment is crucial for prevention of inflammation and autoimmune diseases and is conducted by special immunocompetent cells like macrophages or immature dendritic cells. We recently demonstrated that endothelial cells (ECs) also participate in apoptotic cell clearance. However, in contrast to conventional phagocytes they respond with an inflammatory phenotype. To further confirm these pro-inflammatory responses human ECs were exposed to apoptotic murine ECs and changes in thrombospondin-1 (TSP-1) expression and in activation of intracellular signalling cascades were determined by real-time qPCR, immunoblotting and immunocytochemistry. Human primary macrophages or monocytic lymphoma cells (U937) were incubated with conditioned supernatant of human ECs exposed to apoptotic cells and changes in activation, migration and phagocytosis were monitored. Finally, plasma levels of TSP-1 in patients with anti-neutrophil cytoplasmic antibody(ANCA)-associated vasculitis (AAV) were determined by ELISA. We provided evidence that apoptotic cells induce enhanced expression of TSP-1 in human ECs and that this increase in TSP-1 is mediated by the mitogen-activated protein kinases (MAPK) ERK1 and 2 and their upstream regulators MEK and B-Raf. We also showed that plasma TSP-1 levels are increased in patients with AAV. Finally, we showed that conditioned supernatant of ECs exposed to apoptotic cells induces pro-inflammatory responses in monocytes or U937 cells and demonstrated that increased TSP-1 expression enhances migration and facilitates engulfment of apoptotic cells by monocyte-derived macrophages or U937 cells. These findings suggest that under pathological conditions with high numbers of uncleared dying cells in the circulation endothelial-derived elevated TSP-1 level may serve as an attraction signal for phagocytes promoting enhanced recognition and clearance of apoptotic cells.

Fig 1

Apoptotic cells induce increased TSP-1 expression in ECs. (A) HUVEC or HMEC-1 cells were exposed to apoptotic eEND2 cells for 4 hrs and differences in the expression of TSP-1 transcript was measured by real-time qPCR (n= 8). (B) HUVEC were exposed to apoptotic eEND2 cells for three and for 8 hrs and TSP-1 protein in the supernatant was measured by ELISA (n= 4). (C) Pre-incubation with cytochalasin D did not prevent apoptotic-cell-induced increase in TSP-1 mRNA expression in HUVEC (n= 3). Data are given as mean ± S.D. (*P < 0.05 **P < 0.001).

Fig 2

Apoptotic cell-induced expression of TSP-1 in HUVEC is mediated by the B-Raf/MEK/ERK pathway. (A) Exposure of HUVEC to apoptotic eEND2 cells led to a rapid activation of ERK1/2 (left) and B-Raf (right). Changes in phosphorylation were determined by calculation of the ratio of phosphorylated to unphosphorylated ERK1/2 or B-Raf protein, respectively (n= 6). (B) Immunocytochemical staining of pERK1/2 and pB-Raf in HUVEC exposed to apoptotic eEND2 cells. Cells were counterstained with phalloidin-Alexa546 and analysed by confocal microscopy. Shown are representative micrographs of three independent experiments. Scale bars represent 50 μm. (C) Inhibition of ERK phosphorylation by U0126 or PD98059 circumvented apoptotic cell-induced enhanced TSP-1 expression in HUVEC as assessed by real-time qPCR measurement (n= 4; **P < 0.001).

Fig 3

HUVEC-derived TSP-1 is bound by apoptotic murine eEND2 cells. (A, B) Apoptotic murine eEND2 cells were incubated with conditioned supernatant of HUVEC exposed to apoptotic cells and stained for human TSP-1. Shown are representative confocal images of four independent experiments. The nucleus was counterstained with DAPI. Scale bar represents 10 μm. (C, D) HUVEC were exposed to apoptotic eEND2 cells for 30 min. Cells were counterstained with haematoxylin and eosin and analysed by conventional microscopy. (E, F) HUVEC were seeded onto μ-slides and exposed to apoptotic eEND2 cells for 30 min. Cells were stained for human TSP-1 and applied to confocal microscopy. The nucleus was counterstained with DAPI. Scale bars represent 50 μm.

Fig 4

Supernatant of HUVEC exposed to apoptotic eEND2 cells activates monocyte-derived macrophages and U937 cells. (A) Human monocyte-derived macrophages were exposed to supernatant of HUVEC exposed to apoptotic eEND2 cells, to supernatant of untreated HUVEC or to rh M-CSF for 3 hrs and expression level of different cytokines and chemokines were determined by real-time qPCR. Plotted are the average expression levels of three experiments with macrophages derived from different donors. Note the different y-scale for expression of IL-8. (B) U937 mRNA expression profiles of the same set of cytokines/chemokines after stimulation for 3 hrs with supernatant of untreated HUVEC, HUVEC exposed to apoptotic cells and PMA (n= 3). Note the different y-scale for expression level of MCP-1, TNF-α and IL-10. GoI: Gene of interest.

Fig 5

TSP-1 mediates engulfment of apoptotic cells by macrophages but not by ECs. (A) Blocking TSP-1 in the supernatant with a neutralizing anti-TSP-1 antibody (5 μg/ml) or pre-treatment of HUVEC with RGD-containing peptides (50 μM) or anti-CD36 antibodies (10 μg/ml) did not influence binding and engulfment of apoptotic eEND2 cells. (n= 7). For each experiment at least 10 visual fields were counted and averaged. (B) Blocking RGD-sensitive receptors of monocyte-derived macrophages and PMA-stimulated U937 cells or neutralizing TSP-1 in the conditioned supernatant of apoptotic cell-activated HUVEC reduced the number of engulfed apoptotic cells whereas exogenous TSP-1, added to supernatant of untreated HUVEC, slightly increased the engulfment of apoptotic eEND2. Shown are the results of four to five independent experiments (*P < 0.05; **P < 0.001 compared to pre-incubation with conditioned supernatant alone).

Fig 6

Migration of macrophages or U937 cells is influenced by TSP-1. HUVEC were seeded into the lower compartment of transwell devices and exposed to apoptotic eEND2 cells for 8 hrs or left untreated. Fluorescence-labelled macrophages or U937 cells were added to the upper compartment. In some experiments macrophages or U937 cells were pre-incubated with RGD peptides (50 μM) or TSP-1 in the supernatant was blocked by neutralizing antibodies (5 μg/ml). After 60 min. transmigration of macrophages or U937 cells through the transwell membrane was counted using fluorescence microscopy. Results are given as mean ± S.D. of at least three independent experiments (*P < 0.05).

Fig 7

Plasma TSP-1 concentrations are increased in patients with AAV. Platelet-poor plasma from 19 patients with AAV and from nine healthy volunteers were collected and assayed for TSP-1 concentration using the TSP-1 Quantikine ELISA kit. Results are given as mean ± S.E.M. (*P < 0.005).