Cytokine modulation of liver annexin 1 expression during experimental endotoxemia

Abstract

Annexin 1 (ANXA1) is a calcium-binding protein endowed with anti-inflammatory properties. Using an extra-hepatic system, we showed that interleukin (IL)-6 regulates ANXA1 expression at the transcriptional level. The purpose of this study was to determine whether ANXA1 synthesis was modulated by IL-6 during experimental inflammation. We have compared liver ANXA1 expression during systemic and localized inflammatory reaction, using lipopolysaccharide (LPS) and turpentine. LPS treatment strongly induced ANXA1 expression in the liver of wild-type (WT) animals (+600%) whereas a modest increase (+60%) was measured in IL-6 knockout (KO) animals. Turpentine treatment did not affect the expression of ANXA1 in either animal type. LPS enhanced serum corticosteroid levels equally in WT and IL-6 KO mice, whereas higher tumor necrosis factor (TNF)-alpha and IL-1beta levels were released in IL-6 KO animals. Injection of mouse recombinant IL-6 to IL-6 KO animals before LPS or TNF-alpha challenge, replenished ANXA1 liver synthesis to that of WT animals. Exogenous ANXA1 but not ANXA5, administered to IL-6 KO mice before LPS challenge inhibited TNF-alpha release. We propose that ANXA1 acts as a novel acute phase protein, which is controlled in the liver by TNF-alpha and IL-6, and which may contribute to the resolution of systemic endotoxemia through a negative feedback on TNF-alpha release.

Figure 1.

ANXA1 expression in liver of LPS- or turpentine-challenged animals. A: Constitutive ANXA1 protein expression (37 kd) in the liver of WT and IL-6 KO mice as analyzed by Western blot (see Materials and Methods). The immunoblot was stripped and reprobed with anti-α-tubulin mAb (55 kd). Two animals per group are shown, although data are representative of at least five experiments with similar results. B: Time course of ANXA1 protein expression in WT and IL-6 KO mice after LPS (1 mg/kg intraperitoneally) treatment. C: The same after turpentine (100 μl, intramuscularly) treatment. Densitometric analysis was performed using an Ultrascan XL laser densitometer (Agfa). D: Northern blot analysis of the time course for ANXA1 mRNA expression in the liver of WT and IL-6 KO mice after LPS (1 mg/kg body intraperitoneally). mRNA is expressed as percent increase of naïve animals normalized to GAPDH mRNA expression. Data for the Western blot are expressed as arbitrary units normalized against tubulin, and are shown as mean values ± SEM, n = 10 mice, whereas for Northern blot analysis, histograms are from three animals per group. *, P < 0.01 versus WT.

Figure 2.

Effect of LPS and turpentine on serum CCS, IL-1β, and TNF-α levels in WT and IL-6 KO mice. Mice received 1 mg/kg LPS or 10 ml/kg sterile saline at time 0. CCS levels (A), IL-1β (B), and TNF-α (C) levels in the serum as measured by radioimmunoassay and ELISA, respectively. Data are shown as mean values ± SEM, n = 6. *, P < 0.01 versus appropriate value at time 0. ^§, P < 0.01 versus correspondent WT value.

Figure 3.

ANXA1 immunolocalization in liver of WT or IL-6 KO mice treated with LPS. a and b: WT and IL-6 KO liver sections showed no or scarce ANXA1 expression. c and d: Intense ANXA1 immunostaining is detected in both animal types 4 hours after LPS (1 mg/kg intraperitoneally) injection. All sections were counterstained with Mayer’s hematoxylin. Scale bars, 450 μm.

Figure 4.

Modulation of hepatic ANXA1 protein expression by IL-6 and TNF-α. Western blot analysis of ANXA1 liver expression after treatment with mouse recombinant IL-6 (1 μg i.v.) or TNF-α (1 μg i.v.), alone or in combination with LPS (1 mg/kg intraperitoneally). Protein extracts were prepared as described in Material and Methods. Densitometric analysis was performed using an Ultrascan XL laser densitometer (Agfa). Values represent percent of ANXA1 expression relative to respective controls (WT or IL-6 KO mice). Data are shown as mean values ± SEM, n = 6. *, P < 0.05 and **, P < 0.01 versus relative control (LPS- or TNF-α-treated animals).

Figure 5.

IL-6 and TNF-α up-regulate ANXA1 expression in isolated hepatocytes. Primary hepatocytes were obtained by liver perfusion from WT or IL-6 KO animals as described in Material and Methods. Cells were then treated with IL-6 (10 ng/ml) or TNF-α (2 ng/ml) for the reported time points. Proteins were extracted and Western blot performed according to Material and Methods. A: A representative Western blot is reported for cells taken from both WT and IL-6 KO animals. B: Data (normalized against α-tubulin expression) expressed as percent of untreated hepatocytes (control) are shown as mean values ± SEM, from six different WT or IL-6 KO-perfused liver preparations. *, P < 0.05 versus relative control (unstimulated cells).

Figure 6.

Inhibition of ANXA1 protein expression by neutralizing anti-IL-6 and anti-TNF-α antibodies. WT or IL-6 KO mice were treated by neutralizing antibodies (10 μg intraperitoneally) raised against TNF-α or IL-6 1 hour before LPS (1 mg/kg intraperitoneally) administration. RU-486 (20 mg/kg intraperitoneally) was administrated simultaneously with LPS. Total proteins were extracted from livers and ANXA1 expression was evaluated by Western blot. Histogram represents ANXA1 expression (arbitrary unit) in WT or KO animals, and are representative of three independent experiments each performed with four mice per group. *, P < 0.01.

Figure 7.

Extracellular role of ANXA1 in endotoxemic IL-6 KO mice. IL-6 (1 μg i.v.), human recombinant ANXA1 (10 μg i.v.), or ANXA5 (10 μg i.v.) were administrated 1 hour before LPS (1 mg/kg intraperitoneally) challenge. A: Circulating TNF-α levels were measured by ELISA at the 90-minute time point. B: Circulating IL-1β was measured by ELISA at 4-hour time point. Data are shown as mean values ± SEM, n = 6. *, P < 0.01 versus LPS alone.

Figure 8.

Proposed mechanism for liver ANXA1 expression in experimental endotoxemia. LPS triggers the release of proinflammatory cytokines, including TNF-α, IL-6, and IL-1β that promote expression of ANXA1 in the liver. CCS plays a permissive role. Higher ANXA1 expression, if released in the circulation, may exert a negative feedback on TNF-α and IL-1β release.