Depletion of liver and splenic macrophages reduces the lethality of Shiga toxin-2 in a mouse model

Abstract

The haemolytic uraemic syndrome (HUS) is a clinical syndrome consisting of haemolytic anaemia, thrombocytopenia, and acute renal insufficiency. HUS is the most frequent cause of acute renal failure in childhood. It has been previously suggested that the presence of Shiga toxin (Stx) is necessary but not sufficient for HUS development, and cytokines such as tumour necrosis factor-alpha (TNF-alpha) and IL-1beta appear to be necessary to develop the syndrome. Since the mononuclear phagocytic system (MPS) is the major source of these cytokines, macrophages might be one of the relevant targets for Stx action in the pathophysiology of HUS. In this study our objective was to examine the role of the hepatic and splenic macrophages in a mouse model of HUS induced by injection of Shiga toxin type-2 (Stx2) or Stx2 plus lipopolysaccharide (LPS). For this purpose, depletion of mice macrophages by liposome-encapsulated clodronate (lip-clod), followed by injection of STx2 or Stx2 plus LPS, was assayed. In this study we show that depletion of hepatic and splenic macrophages by clodronate treatment induces a survival of 50% in animals treated with Stx2 alone or in presence of LPS. This maximal effect was observed when lip-clod was injected 48-72 h before Stx2 injection. Biochemical and histological parameters show characteristics of the lesion produced by Stx2, discarding non-specific damage due to LPS or lip-clod. In addition, we determined that the toxic action of Stx2 is similar in BALB/c and N:NIH nude mice, indicating the T cell compartment is not involved in the Stx2 toxicity. Briefly, we demonstrate that macrophages play a central role in the pathophysiology of HUS, and that the systemic production of cytokines by liver and/or spleen is for Stx2 to manifest its full cytotoxic effect. In addition, the toxicity of Stx2 alone, or in presence of LPS, is independent of the T cell compartment.

Fig. 1

Liposome-encapsulated clodronate (lip-clod) treatment impaired the kinetics of ⁵¹Cr-EA disappearance from circulation. The Figure represents the survival of ⁵¹Cr-labelled IgG-sensitized erythrocytes (EA) in saline (•—–•) and lip-clod-treated mice (•- - - - -•). Unsensitized erythrocytes (E) in saline (○—–○) and lip-clod-treated mice (○- - - - -○) are also depicted. BALB/c mice were intravenously injected with 200 μl of lip-clod 72 h before E or EA injection. Each point represents mean ± s.e.m. of at least six animals. Circulating EA survival of lip-clod-treated group at 24 h was significantly different from that of the saline group (P < 0.001) by Bonferroni's t-test.

Fig. 2

Tissue distribution of ⁵¹Cr-labelled IgG-sensitized erythrocytes (EA) in liposome-encapsulated clodronate (lip-clod)-treated mice. BALB/c mice were intravenously injected with 200 μl of lip-clod 72 h before E or EA injection. After 24 h of E or EA injection mice were killed and their organs excised. Each bar represents the mean of the percentage of the radioactivity sequestered ± s.e.m. of six animals. Organ uptake of unsensitized E in saline and lip-clod-treated mice is also depicted and no significant differences between groups were observed. *P < 0.001 compared with saline EA by Bonferroni's t-test.

Fig. 3

Liposome-encapsulated clodronate (lip-clod) treatment partially inhibits Shiga toxin type 2 (Stx2) and Stx2 + lipopolysaccharide (LPS) lethality. BALB/c mice (24 mice/group, which corresponds to two independent experiments with 12 mice/group each) were intravenously injected with 250 pg of Stx2 (•—–•) or intraperitoneally injected with 5 μg of LPS 1 h before injection of 250 pg of Stx2 (○—–○). Two additional groups of mice were previously treated with 200 μl of lip-clod, and 72 h later were intravenously injected with Stx2 (•- - - - -•) or LPS + Stx2 (○- - - - -○) in the same conditions as described. The death of animals was followed up to 200 h after Stx2 injection, times in which there were no additional mortalities than those shown in the Figure. *P < 0.05 compared with Stx2-treated mice at 84 h; **P < 0.01 compared with Stx2-treated mice at 84 and 108 h; †P < 0.001 compared with (LPS − 1 h + Stx2)-treated mice at 84 and 108 h, evaluated by Fisher's exact test.

Fig. 4

Time-dependence of liposome-encapsulated clodronate (lip-clod) injection on Shiga toxin type 2 (Stx2) toxicity. Twelve BALB/c mice per group were intravenously injected with 200 μl of lip-clod at the times indicated in the Figure. Stx2 (250 pg/mouse) was intravenously injected at 0 h. The death of animals was followed up to 200 h after Stx2 injection. Solid line represent the control (Stx2, 250 pg/mouse). *P < 0.01 compared with Stx2-treated mice.

Fig. 5

Shiga toxin type 2 (Stx2) action on T cell-deficient mice. Groups of 10 BALB/c and 10 N:NIH nude mice were intravenously injected with 250 pg of Stx2 or 5 μg of lipopolysaccharide (LPS), and 1 h later 250 pg Stx2 (Stx2 + LPS). Differences between groups were not significant.