Published online before print November 10, 2006
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* Departamentos de Bioquímica e Imunologia and
Microbiologia, ICB, Universidade Federal de Minas Gerais, Belo Horizonte, Minas Gerais, Brazil; and
Division of Immunology, Infection and Inflammation, University of Glasgow, Glasgow, UK
1 Correspondence: Departamento de Bioquímica e Imunologia, Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais, Av. Antonio Carlos, 6627–Pampulha, 31270-901 Belo Horizonte, MG, Brazil. E-mail: mmtex{at}mono.icb.ufmg.br
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dose-dependently in the intestine and in the lungs. The sST2-Fc treatment with the highest dose (100 µg) resulted in inhibited vascular permeability, neutrophilia, and hemorrhage in the intestine and the lungs compared with controls treated with normal IgG. This was associated with down-regulated tissue levels of proinflammatory cytokines, markedly reduced serum TNF- levels, and increased survival of mice from the sST2-Fc-treated group after ischemia and reperfusion injury. The beneficial effect of sST2-Fc treatment was associated with elevated IL-10 production in intestine and lung. sST2-Fc was not able to prevent the inflammatory response associated with intestinal ischemia and reperfusion in IL-10-deficient mice, suggesting that sST2 exerts its anti-inflammatory effect in a IL-10-dependent manner. These results also demonstrate that sST2-Fc may provide a novel, complementary approach in treating ischemic reperfusion injury.
Key Words: neutrophil influx • IL-10 • TNF-
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ST2 gene expression was identified originally by serum stimulation in fibroblasts [12
13
14
]. It was found to be expressed subsequently in mast cells and Th2 but not Th1 cells [15
16
17
18
]. Structurally, ST2 is a member of the IL-1 receptor (IL-1R) family; however, it does not bind IL-1, IL-1ß, or IL-1R antagonist, and its functional ligand is unknown [19
, 20
]. The mRNA processing of the ST2 gene leads to the production of three different products: a soluble-secreted form (sST2), expressed mainly in fibroblasts; a membrane-bound receptor form (ST2L), expressed primarily in hemopoietic cells [21
]; and a variant form [22
], expressed in humans. sST2 is identical to the extracellular region of ST2L, except for nine amino acids present at the C-terminal of ST2. The transcription of the ST2 gene is controlled by two distinct promoters: An upstream promoter controls the transcription in hemopoietic cells, and a promoter 10.5 kb downstream directs fibroblast-specific expression [21
].
ST2 is involved in the regulation of Th1/Th2-associated immune responses [17
18
19
20
21
22
23
24
25
]. Recently, we have demonstrated that ST2–/– mice produced elevated concentration of proinflammatory cytokines and were unable to develop endotoxin tolerance [26
]. Elevated sST2 production has been reported in inflammatory conditions, such as UVB irradiation-induced inflammation and LPS-induced lung acute inflammation [27
28
29
30
]. It is interesting that sST2 expression is also induced after myocardial infarction in mice [31
]. Moreover, sST2-Fc fusion protein suppressed the production of TNF-, IL-6, and IL-12 and mortality induced by LPS in vivo in mice [32
]. sST2-Fc fusion protein administration also had a beneficial effect in a murine model of collagen-induced arthritis [33
].
We report here that sST2-Fc fusion protein administration markedly reduced local, remote, and systemic, proinflammatory cytokine production and tissue injury after reperfusion of the superior mesenteric artery in mice. In addition, sST2-Fc significantly increased IL-10 concentration induced by ischemia and reperfusion. These events culminate in significant attenuation of lethality in treated mice. It is interesting that the anti-inflammatory effect exerted by sST2-Fc was dependent on the production of IL-10, as demonstrated by experiments in IL-10-deficient mice. Our results, therefore, suggest that sST2-Fc fusion protein may have considerable therapeutic potential as an adjunct to the treatment of ischemic events.
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Reagents
Urethane, Evans blue, tetramethylbenzidine, and hexadecyltrimethylammonium bromide were obtained from Sigma Chemical Co. (St. Louis, MO). The sST2-Fc fusion protein was prepared as described previously [32
]. Briefly, a mammalian expression plasmid containing the sST2 cDNA linked to the human IgG1 constant region and containing the C5 signal sequence was transfected into Chinese hamster ovary cells. The sST2-Fc was purified using a protein affinity chromatography. Purified sST2-Fc was found to contain <0.1 ng/µg LPS, as assessed by the Limulus amoebocyte assay (Sigma-Aldrich, Cambridge, UK).
Ischemia and reperfusion
Mice were anesthetized with urethane (1400 mg/kg, i.p.), and laparotomy was performed. Superior mesenteric artery (SMA) was isolated, and ischemia was induced by total occlusion for 60 min. For measuring the percentage of surviving mice, reperfusion was re-established, and mice were monitored for indicated time periods. For the other parameters, reperfusion was allowed to occur for 30 min (I60R30) when mice were killed. This time of reperfusion (30 min) was chosen based on the presence of significant tissue injury without unduly high mortality rates. Sham-operated animals were used as controls. sST2-Fc fusion protein (1, 10, and 100 µg/mouse) or control, normal IgG was administrated i.v., 10 min before reperfusion.
Evaluation of changes in vascular permeability
The extravasation of Evans blue dye into the tissue was used as an index of increased vascular permeability, as described previously [34
, 35
]. Evans blue (20 mg/kg) was administered i.v. (1 ml/kg) via a tail vein, 2 min prior to reperfusion of the ischemic artery. After 30 min reperfusion, a segment of the duodenum (
3 cm) was cut open and allowed to dry in a Petri dish for 24 h at 37°C. The dry weight of the tissue was calculated, and Evans blue was extracted using 1 ml formamide (24 h at room temperature). The amount of Evans blue in the tissue was obtained by comparing the extracted absorbance with that of a standard Evans blue curve read at 620 nm in an ELISA plate reader. Results are presented as the amount of Evans blue/µg/100 mg tissue. The right ventricle was flushed with 10 ml PBS to wash the intravascular Evans blue in the lungs. The left lung was then excised and used for Evans blue extraction as described above. The right lung was used for the determination of myeloperoxidase (MPO) as described below.
MPO concentrations
The extent of neutrophil accumulation in the intestine and the lungs was measured by assaying MPO activity as described previously [8
, 36
]. Briefly, a portion of the duodenum and the flushed-right lungs of animals that had undergone ischemia/reperfusion injury was removed and snap-frozen in liquid nitrogen. Upon thawing and processing, the tissue was assayed for MPO activity by measuring the change in OD at 450 nm using tetramethylbenzidine. Results were expressed as the neutrophil infiltration. An index unit denotes the MPO activity present in 105 casein-elicited, murine peritoneal neutrophils processed in the same way.
Measurement of hemoglobin concentrations
Hemoglobin concentration in tissue was used as an index of tissue hemorrhage. After washing the intestines to remove excess blood, a sample of 100 mg duodenum was removed and homogenized in Drabkin’s color reagent according to the manufacturer’s instructions (Analisa, Belo Horizonte, Brazil). The suspension was centrifuged for 15 min at 3000 g and filtered using 0.2 µm filters. The resulting solution was read using an ELISA plate reader at 520 nm and compared against a standard curve of hemoglobin.
Measurement of cytokine/chemokine concentrations in serum, intestine, and lungs
The concentration of TNF-, IL-10, CXCL1, and CCL2 in samples was measured in serum and tissues of animals using commercially available antibodies and according to the procedures supplied by the manufacturer (R&D Systems, Minneapolis, MN). Serum was obtained from coagulated blood (15 min at 37°C and then 30 min at 4°C) and stored at –20°C until analysis. Samples were analyzed at 1:3 dilutions in PBS. Duodenum (100 mg) or lung of sham-operated and reperfused animals was homogenized in 1 ml PBS (0.4 M NaCl and 10 mM NaPO4) containing antiproteases (0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA, and 20 KI aprotinin A) and 0.05% Tween 20. The samples were then centrifuged for 10 min at 3000 g, and the supernatant was used immediately for ELISA assays at 1:3 dilution in PBS.
Histopathology
Sections of the duodenum were obtained from similar areas of the small intestine from anesthetized mice at indicated time-points and fixed immediately in 10% formalin for 24 h, and tissues fragments were embedded in paraffin. Tissue sections (4 µm thick) were stained with H&E and examined under light microscope. Lungs were inflated with 2 ml 10% buffered formalin, removed from anesthetized animals, and embedded and sectioned as above.
Statistical analysis
Results are shown as means ± SEM. As there was no difference between sST2- and IgG-treated animals in the baseline values of the parameters studied, results were pooled for ease of presentation and are shown in the figures as "sham." Percent inhibition was calculated by subtracting the background values obtained in sham-operated animals. Differences were compared by using ANOVA followed by Student-Newman-Keuls post hoc analysis. P < 0.05 was considered significant.
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production dose-dependently after intestinal ischemia and reperfusion in different inflammatory conditions [32
, 33
]. Initial experiments were designed to assess the effects of sST2 treatment in the TNF- production in intestine and lungs of mice subjected to intestinal ischemia and reperfusion. The murine model of ischemic reperfusion was performed as described in Materials and Methods, and sST2-Fc (1, 10, and 100 µg/animal) or control IgG was injected i.v., 10 min prior to the reperfusion. After the reperfusion, an exacerbated production of this cytokine was observed in both organs of control, IgG-treated animals. However, the TNF- levels were diminished in intestine and lungs of the sST2-Fc-treated group. This inhibition of TNF- by sST2-Fc happened in a dose-dependent manner, and the maximal effect of the treatment was achieved with the dose of 100 µg/animal (Fig. 1
). This dose was chosen for all subsequent experiments.
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Figure 1. Effects of treatment with sST2-Fc on TNF- concentrations in the intestine (a) and lungs (b) in a model of intestinal ischemia (60 min) and reperfusion (30 min) injury (I60R30). The concentrations of TNF- in both organs were determined by ELISA. sST2-Fc (1, 10, or 100 µg/mouse) or control, normal IgG was given i.v., 10 min prior to reperfusion. As there was no difference between sST2-Fc- and IgG-treated animals in the baseline values of TNF-, results were pooled for ease of presentation and are shown in the figure as sham. Results are shown as pg/100 mg tissue and are mean ± SEM of six animals. *, P < 0.01, compared with sham-operated animals; #, P < 0.01, compared with IgG-treated mice. ND, Not determined.
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production locally and remotely (Fig. 1)
, our next step was to evaluate whether sST2-Fc treatment would be able to inhibit ischemia reperfusion-induced injuries. As observed in Figure 2 , after reperfusion of the ischemic SMA, intestine (Fig. 2a) and lungs (Fig. 2b) of control, IgG-treated mice presented a great rise in neutrophil influx to tissue. This was supported by an elevation of the chemokine CXCL1 and CCL2 levels in both organs. The animals that received sST2-Fc treatment before reperfusion had a significant reduction in neutrophil influx and chemokine production in both organs (Fig. 2a and 2b) . The intensity of neutrophil recruitment after intestinal ischemia and reperfusion was correlated with tissue injury, as assessed by an increase in vascular permeability and hemorrhage in intestine and lungs. Hence, the vascular permeability was elevated in both organs of the control, IgG-treated group (Fig. 3a and 3b ). The levels of intestinal hemorrhage, accessed by hemoglobin levels, were also altered (Fig. 3c) . These alterations were absent in the sST2-Fc-treated group (Fig. 3a 3b 3c) .
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Figure 2. Effects of treatment with sST2-Fc on the neutrophil influx, CXCL1, and CCL2 concentrations in the intestine (a) and lung (b) in a model of intestinal ischemia (60 min) and reperfusion (30 min) injury (I60R30). The neutrophil migration was assessed by measuring the tissue contents of MPO, and CXCL1 and CCL2 concentrations were determined by ELISA. sST2-Fc (100 µg/mouse) or IgG was given i.v., 10 min prior to reperfusion. As there was no difference between sST2-Fc- and IgG-treated animals in the baseline values of neutrophils, CXCL1, and CCL2, results were pooled for ease of presentation and are shown in the figure as sham. Results are shown as number of neutrophils/100 mg tissue and pg/100 mg tissue and are mean ± SEM of six animals. *, P < 0.05, compared with sham-operated animals; #, P < 0.05, compared with IgG-treated mice.
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Figure 3. Effects of treatment with sST2-Fc on the changes in vascular permeability in the intestine (a) and lung (b) and on the intestinal hemoglobin content (c) in a model of intestinal ischemia (60 min) and reperfusion (30 min) injury (I60R30). Changes in vascular permeability were assessed by measuring Evans blue dye extravasation, and tissue hemorrhage was assessed by evaluating the tissue levels of hemoglobin. sST2-Fc (100 µg/mouse) or IgG was given i.v., 10 min prior to reperfusion. Results are mean ± SEM of six animals. *, P < 0.05, compared with sham-operated animals; #, P < 0.05, compared with IgG-treated mice.
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Figure 4. Effects of treatment with sST2-Fc on tissue damage in intestine (a–c) and lung (d–f) in a model of intestinal ischemia and reperfusion injury. Mice were sham-operated (a, d) or submited to 60 min of ischemia of the SMA, and reperfusion was allowed for 30 min. Control IgG (b, e) or sST2-Fc (c, f) was given i.v., 10 min prior to reperfusion. Mice were anesthetized and killed, and intestine and lungs were processed for histological analysis after H&E staining. Original magnification, x20.
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and survival following ischemia and reperfusion [10
]. We then determined the effect of sST2-Fc treatment on the survival and serum TNF- concentrations in the reperfused animals. Reperfused mice treated with sST2-Fc reduced serum concentrations of TNF- strikingly when compared with control, IgG-treated mice (Fig. 5a
). Concomitantly, sST2-Fc administration significantly attenuated the lethality of ischemic reperfusion. All control, IgG-treated animals died after 120 min of reperfusion, whereas 50% of sST2-Fc-treated animals survived the reperfusion (Fig. 5b)
.
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Figure 5. Effects of treatment with sST2-Fc on the serum concentrations of TNF- and mortality in a model of intestinal ischemia (60 min) and reperfusion (30 min) injury (I60R30). TNF- (a) was measured by ELISA. Survival (b) was monitored as indicated. sST2-Fc (100 µg/mouse) or control IgG was given i.v., 10 min prior to reperfusion. TNF- was not detected in serum of sham-operated mice given sST2-Fc or IgG (shown as sham). Results are mean ± SEM of five to six (a) or eight to 11 (b) mice. *, P < 0.05, compared with sham-operated animals; #, P < 0.05, compared with IgG-treated mice.
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Figure 6. Effects of treatment with sST2-Fc on the concentrations of IL-10 in the intestine (a) and lung (b) in a model of intestinal ischemia (60 min) and reperfusion (30 min) injury (I60R30). IL-10 concentrations were assessed by ELISA. sST2-Fc (100 µg/mouse) or IgG was given i.v., 10 min prior to reperfusion. As there was no difference between sST2-Fc- and IgG-treated animals in the baseline values of IL-10, results were pooled for ease of presentation and are shown in the figure as sham. Results are mean ± SEM of six animals. *, P < 0.05, compared with sham-operated animals; #, P < 0.05, compared with IgG-treated mice.
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production and neutrophil influx in IgG or sST2-Fc-treated IL-10–/– animals, as shown in Table 1
. Similarly, tissue damage, as assessed by plasma extravasation and hemorrhage, and local production of chemokines, as assessed by CXCL1 measurements, were equivalent in Ig and sST2-Fc-treated IL-10–/– mice. There was also a similar elevation in the concentration of TNF- in serum of IL-10–/– mice treated with Ig or sST2-Fc. These results indicate that the protective effect of sST2-Fc administration in this model is IL-10-dependent. |
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Table 1. Effects of Treatment with sST2-Fc on the Injury Induced by Intestinal Ischemia (60 min) and Reperfusion (30 min) in IL-10-Deficient Mice
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The beneficial effect of sST2 treatment in our model is associated closely with the profound reduction in the production of proinflammatory mediators. Significantly, sST2 treatment reduced serum TNF- levels markedly, the elevation of which is associated closely with ischemic reperfusion injury [10
, 39
40
41
]. Consistent with this anti-inflammatory effect, we also observed an inhibition of the expression of CCL2 and CXCL1 in mice that received sST2-Fc treatment. CCL2 and CXCL1 are well-characterized mediators of leukocyte migration and are found in elevated concentrations in serum and tissue after ischemia and reperfusion [42
, 43
]. Moreover, we have shown recently that blockade of CXCR2, the receptor for CXCL1 in mice, significantly prevented reperfusion injury in rodents [5
]. Hence, the low concentrations of these chemokines may have contributed to the reduced injury in the intestine and the lungs by decreasing the influx of neutrophils. Our previous studies have suggested the critical role of TNF- in mediating the infiltration of neutrophils in the duodenum and lungs of reperfused animals. Consequently, the inhibition of neutrophil influx may be secondary to inhibition of CXCL1 and TNF- by sST2 treatment. sST2 could potentially down-regulate the expression of TLR1 and TLR4 [32
], the signaling of which is important in the induction of a range of proinflammatory cytokines [44
]. Nevertheless, we have reported previously that tissue injury and lethality, following reperfusion of the ischemic superior mesenteric artery, was not dependent on TLR4 [4
]. Thus, this is not the precise mechanism by which sST2 inhibits the production of the inflammatory mediators in the intestinal ischemia and reperfusion model.
It is interesting that sST2 treatment in the current model also led to increased production of IL-10, an established anti-inflammatory cytokine. Earlier reports have shown that IL-10 modulated proinflammatory cytokine production and tissue injury after ischemia and reperfusion [37
, 38
]. Recently, our group demonstrated that exogenous administration of IL-10 reduced the systemic inflammation in rats during intestinal ischemia and reperfusion and prevented the increase in tissue and serum TNF- concentrations. This was associated with reduced injury and lethality. Moreover, treatment with anti-IL-10 antibody was associated with increased TNF- synthesis, tissue injury, and lethality, demonstrating an important role of endogenous IL-10 in this system [7
]. Thus, the ability of sST2 to provoke enhancement of the synthesis of IL-10 could in turn lead to an inhibition of proinflammatory cytokine synthesis, reduced neutrophilia, and attenuated tissue destruction and lethality. Our previous studies have found that sST2 did not inhibit the production of IL-10 by LPS-stimulated, bone marrow-derived macrophages, but a stimulatory effect was not noticed [32
]. To clarify if the anti-inflammatory activity of sST2 in the model was dependent on IL-10, we conducted experiments in IL-10–/– mice. Hence, sST2 administration, before reperfusion of IL-10–/– mice, was unable to reduce the inflammatory response and tissue injury, supporting the hypothesis that the beneficial effects of sST2 in the system are secondary to its IL-10-enhancing capabilities. The mechanisms by which sST2 exerts this activity are currently unclear. sST2 could potentially be acting on macrophages to enhance reperfusion-induced IL-10 production. Alternatively, as IL-10 is associated closely with the suppressive activities of regulatory T cells (Treg) [45
], the possibility that sST2 activates populations of Treg cells preferentially merits further investigation.
In conclusion, we demonstrated here that sST2-Fc treatment reduced tissue injury and mortality significantly in a murine model of ischemia and reperfusion. This activity is a result of elevation of IL-10 synthesis and consequent inhibition of production of proinflammatory mediators. Our results provide direct evidence for a causal association between sST2 and vascular injury. These data also suggest that sST2-Fc fusion protein is a potential, complementary, therapeutic agent against ischemia reperfusion injury.
Received June 30, 2006; revised August 2, 2006; accepted August 21, 2006.
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on local and remote injuries following ischaemia and reperfusion injury Br. J. Pharmacol. 134,985-994[CrossRef][Medline] and interleukin-1ß in ischemia-reperfusion injury of the rat small intestine J. Surg. Res. 99,134-141[CrossRef][Medline] and interleukin-1 in limb ischemia/reperfusion injury and associated lung injury Am. J. Pathol. 143,453-463[Abstract]
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