High mobility group box 1 and adenosine are both released by endothelial cells during hypothermic preservation

Abstract

Hypothermic preservation of solid allografts causes profound damage of vascular endothelial cells. This, in turn, might activate innate immunity. In the present study we employed an in vitro model to study to what extent supernatants of damaged endothelial cells are able to activate innate immunity and to study the nature of these signals. The expression of high mobility group box 1 (HMGB1) and adhesion molecules on human umbilical vein endothelial cell was studied by immunofluorescence, fluorescence activated cell sorter and Western blotting. Cytokine production was performed by enzyme-linked immunosorbent assay. HMGB1 expression was lost completely in endothelial cells after hypothermic preservation. This was associated with cell damage as it occurred only in untreated endothelial cell but not in cells rendered resistant to hypothermia-mediated damage by dopamine treatment. Only supernatants from hypothermia susceptible cells up-regulated the expression of interleukin (IL)-8 and adhesion molecules in cultured endothelial cells in an HMGB1-dependent manner. In whole blood assays, both supernatants of hypothermia susceptible and resistant cells inhibited tumour necrosis factor (TNF)-alpha production concomitantly with an increased IL-10 secretion. The activity of the supernatants was already found after 6 h of hypothermic preservation, and paralleled the decrease in intracellular adenosine triphosphate (ATP) levels. Modulation of TNF-alpha and IL-10 production by these supernatants was abrogated completely by prior treatment with adenosine deaminase and was similar to the response of an A2R agonist. Our study demonstrates that both HMGB1 and adenosine are released during hypothermic preservation. While release of HMGB1 is caused by cell damage, release of adenosine seems to be related to ATP hydrolysis, occurring in both susceptible and resistant cells.

Fig. 1

(a) High mobility group box 1 (HMGB1) expression in endothelial cells. Cultured human umbilical vein endothelial cells (HUVEC) (upper two panels, left) and freshly isolated peripheral blood mononuclear cells (upper panel, right) were stained for HMGB1 as described in the Materials and methods section. Analysis of HMGB1 was performed by means of immunofluorescence (upper panels, left and right) and by confocal microscopy. Original magnification: 200× and 400× respectively. The lower panels show HMGB1 expression in untreated HUVEC (left) and dopamine-treated HUVEC (right) after 24 h of cold storage. Original magnification: 400×. (b) Western blot analysis of HMGB1 in cell nuclei from HUVEC before (37°C) and after cold storage (4°C). Note the disappearance of HMGB1 from the nuclei after cold storage of untreated HUVEC (–) and its appearance the supernatant of these cells. Da: dopamine 30 μM.

Fig. 2

Up-regulation of adhesion molecules and interleukin (IL)-8 production by supernatants. (a) Normal cultured endothelial cell were incubated for 24 h with supernatants obtained from untreated (bold line) and dopamine-treated (thin line) human umbilical vein endothelial cells (HUVEC) that were subjected to 24 h of cold storage. Note that supernatants from untreated HUVEC up-regulates the expression of intercellular adhesion molecule type 1, E-selectin and vascular cell adhesion molecule type 1 compared with supernatants from dopamine treated HUVEC. Filled histogram represents the negative control. The results from a representative experiment (n = 5) is depicted. (b) Normal cultured endothelial cells were incubated for 24 h with supernatants obtained from untreated (Sup-untreated) and dopamine-treated (Sup-Da-treated) HUVEC that were subjected to 24 h of cold storage. The supernatants were immune-absorbed with anti-high mobility group box 1 (HMGB1) (filled bars) or normal rabbit immunoglobulin G, as described in the Materials and methods section. IL-8 production was measured by enzyme-linked immunosorbent assay using triplicates for each condition. The results are expressed as mean IL-8 production (ng/ml) ± standard deviation from four different experiments. ICAM, intracellular adhesion molecule; VCAM, vascular adhesion molecule.

Fig. 3

Lipopolysaccharide (LPS)-mediated tumour necrosis factor (TNF)-α and interleukin (IL)-10 production in whole blood assays. Whole blood was diluted 1 : 1 in normal phenol-red free (PRF) medium to which either nothing (open bars) or 0·1 μg/ml recombinant high mobility group box 1 (HMGB1) (filled bars) was added. In addition, whole blood was diluted 1 : 1 with supernatants obtained after cold storage (24 h) of untreated human umbilical vein endothelial cells (HUVEC) (Sup-untreated; dotted bars) or dopamine-treated (Sup-Da-treated; hatched bars). To each condition 0·5 μg/ml of LPS was added. TNF-α and IL-10 production was assessed after 24 h by enzyme-linked immunosorbent assay, using triplicates for each condition. A total of six experiments were performed with different blood donors and different HUVEC supernatants. The result of a representative experiment is depicted.

Fig. 4

Modulation of Toll-like receptor (TLR)-mediated tumour necrosis factor (TNF)-α and interleukin (IL)-10 production. Whole blood was diluted 1 : 1 in normal phenol-red free (PRF) medium (open bars) or in supernatants of untreated human umbilical vein endothelial cells (Sup-untreated), obtained directly after 24 h of cold storage. To the wells were added either nothing (no) or different TLR ligands (TLR-1–9). The different ligands that were used were as follows: peptidoglycan Bacillus subtilis (PGN-BS) (20 μg/ml) (TLR-1), Pam3CysSerLys4 (10 μg/ml) (TLR-2), Poly (I:C) (50 μg/ml) (TLR-3), LPS 500 ng/ml (TLR-4), flagellin (1 μg/ml) (TLR-5), CL087 (1 μg/ml) (TLR-6), ssRNA (1 μg/ml) (TLR-8) and ODN2006 (1 μM) (TLR-9). TNF-α and IL-10 production was assessed as described in Fig. 3. A total of four different experiments were performed. The result of a representative experiment is expressed as mean cytokine production (pg/ml) ± standard deviation.

Fig. 6

Modulation of lipopolysaccharide (LPS)-induced cytokine production is mediated by adenosine. (a) Whole blood was diluted 1:1 in normal phenol-red free (PRF) medium (open bars) and in supernatants of untreated human umbilical vein endothelial cells (HUVEC) (filled bars; Sup-untreated) or in supernatants of dopamine treated HUVEC (hatched bars; Sup-Da-treated), obtained directly after 24 h of cold storage. To each condition 0·5 μg/ml of LPS was added. The whole blood assay was performed in the absence (medium) or presence of 5′-N-ethylcarboxamidoadenosine (10 μM). (b) Similar to (a), but the supernatants were not treated (medium) or treated with adenosine deaminase (7 U/ml) before initiation of the experiments. In (a) and (b), tumour necrosis factor (TNF)-α and interleukin (IL)-10 production were assessed as described in Fig. 3. A total of four different experiments were performed. The result of a representative experiment is depicted and expressed as mean cytokine production (pg/ml) ± standard deviation.

Fig. 5

Acquirement of tumour necrosis factor (TNF)-α suppressing activity in supernatants and intracellular adenosine triphosphate (ATP) depletion in time. (a) Whole blood was diluted 1:1 in normal phenol-red free (PRF) medium (filled bars) and in supernatants of untreated human umbilical vein endothelial cells (HUVEC) (Sup-untreated) or in supernatants of dopamine-treated HUVEC (Sup-Da-treated), obtained directly after 6 h (open bars) or 12 h (hatched bars) of cold storage. To each condition 0·5 μg/ml of lipopolysaccharide was added. TNF-α production was assessed as described in Fig. 3. A total of three different experiments were performed. The result of a representative experiment is expressed as mean TNF-α production (pg/ml) ± standard deviation (s.d.). (b) Untreated (open bars) and dopamine-treated (filled bars) HUVEC were stored at 4°C for different periods of time. Hereafter, intracellular ATP was measured as described in Materials and methods. The total amount of ATP before cold storage was taken as 100% for each condition. The result of a representative experiment is depicted and expressed as mean percentage of total ATP ± s.d. A total number of three experiments were performed.