IL-12 suppression, enhanced endocytosis and up-regulation of MHC-II and CD80 in dendritic cells during experimental endotoxin tolerance

Acta Pharmacologica Sinica, Apr 2009

Aim: The aim of this study was to investigate endocytosis, MHC-II expression and co-stimulatory molecule expression, as well as interleukin-12 (IL-12) production, in bone marrow dendritic cells (DCs) derived from endotoxin tolerant mice. Methods: Endotoxin tolerance was induced in C57BL/10J mice through four consecutive daily intraperitoneal injections of 1.0 mg/kg of 055:B5 Escherichia coli lipopolysaccharide (LPS). Bone marrow DCs were isolated in the presence of GM-CSF and IL-4 and purified by anti-CD11c Micro beads. FITC–dextran uptake by DCs was tested by flow cytometric analysis and the percentage of dextran-containing cells was calculated using a fluorescence microscope. The expression of surface MHC-II, CD40, CD80, and CD86 was also detected by flow cytometric analysis. An ELISA was used for the measurement of IL-12 production by DCs with or without LPS stimulation. Results: Endotoxin tolerance was successfully induced in C57BL/10J mice, evidenced by an attenuated elevation of systemic TNF-α. DCs from endotoxin tolerant mice possessed enhanced dextran endocytosis ability. The expression of surface MHC-II and CD80 was higher in DCs from endotoxin tolerant mice than in DCs from control mice, whereas the expression of CD40 and CD86 was not altered. Compared with DCs from normal control mice, DCs from endotoxin tolerant mice produced less IL-12 after subsequent in vitro stimulation with LPS. Conclusion: These data suggest enhanced endocytosis, selective up-regulation of MHC-II and CD80 and IL-12 suppression in DCs during in vivo induction of endotoxin tolerance.

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IL-12 suppression, enhanced endocytosis and up-regulation of MHC-II and CD80 in dendritic cells during experimental endotoxin tolerance

Abstract Aim: The aim of this study was to investigate endocytosis, MHC-II expression and co-stimulatory molecule expression, as well as interleukin-12 (IL-12) production, in bone marrow dendritic cells (DCs) derived from endotoxin tolerant mice. Methods: Endotoxin tolerance was induced in C57BL/10J mice through four consecutive daily intraperitoneal injections of 1.0 mg/kg of 055:B5 Escherichia coli lipopolysaccharide (LPS). Bone marrow DCs were isolated in the presence of GM-CSF and IL-4 and purified by anti-CD11c Micro beads. FITC–dextran uptake by DCs was tested by flow cytometric analysis and the percentage of dextran-containing cells was calculated using a fluorescence microscope. The expression of surface MHC-II, CD40, CD80, and CD86 was also detected by flow cytometric analysis. An ELISA was used for the measurement of IL-12 production by DCs with or without LPS stimulation. Results: Endotoxin tolerance was successfully induced in C57BL/10J mice, evidenced by an attenuated elevation of systemic TNF-α. DCs from endotoxin tolerant mice possessed enhanced dextran endocytosis ability. The expression of surface MHC-II and CD80 was higher in DCs from endotoxin tolerant mice than in DCs from control mice, whereas the expression of CD40 and CD86 was not altered. Compared with DCs from normal control mice, DCs from endotoxin tolerant mice produced less IL-12 after subsequent in vitro stimulation with LPS. Conclusion: These data suggest enhanced endocytosis, selective up-regulation of MHC-II and CD80 and IL-12 suppression in DCs during in vivo induction of endotoxin tolerance. Introduction Lipopolysaccharide (LPS)-containing endotoxins are potent activators of the innate immune system and are central to the pathological excessive or deregulated inflammation observed in Gram-negative sepsis. The endotoxins can lead to systemic inflammatory response syndrome, multiple organ failure and even death1, 2. Therefore, the prevention of excessive release of pro-inflammatory mediators through medications such as corticosteroid and anti-tumor necrosis factor-α (TNF-α) antibody is one of the vital strategies for the prevention and treatment of tissue injury in this clinical setting1, 2, 3. However, none of these treatments showed significant improvement in clinical trials4. In part, the benefit of the suppression of proinflammatory mediators is thought to be counteracted by the all-around inhibition of the immune system. Consecutive low doses of LPS can induce endotoxin tolerance in which endotoxin triggered responses are at least partially abrogated5. In vitro, the endotoxin-tolerant monocytes and macrophages are characterized by inhibition of LPS-stimulated proinflammatory cytokine production5, 6, 7. Our previous study demonstrated that endotoxin-tolerant animals had a marked reduction in the systemic response to endotoxemia, including mortality, weight loss, fever and serum cytokine level8, in accordance with other in vivo studies5, 7, 9, 10, 11. Moreover, several previous studies showed that the repetitive low-dose LPS pretreated mice had improved survival after bacterial and fungal infection12, 13. The fundamental mechanisms that underlie such beneficial effects of endotoxin tolerance have been increasingly studied recently and might be associated with the reprogramming of immune cells during LPS pretreatment. Dendritic cells (DCs) are composed of different subgroups and coordinate the innate and the adaptive arms of the immune response. They are scattered throughout the body, where they work as “immunological sensors” that are capable of decoding the dangerous signals, presenting the information to T cells and generating the right class of immune responses14, 15, 16, 17, 18. Currently, little is known about the variation of DCs during the induction of endotoxin tolerance. To develop a greater understanding of the alteration of DCs during LPS pretreatment, we reproduced murine endotoxin tolerance through repetitive intraperitoneal injections of low dose LPS and investigated three functions of isolated bone marrow DCs: (1) endocytotic capacity; (2) expression of MHC-II and co-stimulatory molecules on the cell surface; and (3) the ability to produce interleukin-12 (IL-12). Materials and methods Animals and treatment Specific pathogen-free male C57BL/10J mice at 4−6 weeks of age were purchased from Shanghai SLAC Laboratory Animal Inc/National Rodent Laboratory Animal Resources, Shanghai branch, and bred under specific pathogen-free conditions in the animal facility of the Liver Cancer Institute of Zhongshan Hospital, Fudan University. The procedures involving animals and their care were conducted in conformity with national and international laws and policies. The mice were randomly divided into the endotoxin tolerant group, which was pretreated with an intraperitoneal injection of 1 mg/kg Escherichia coli LPS (serotype 055:B5; Sigma, St Louis, MO) at the same time on each of four consecutive days, and t (...truncated)


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Jing Zhang, Jie-ming Qu, Li-xian He. IL-12 suppression, enhanced endocytosis and up-regulation of MHC-II and CD80 in dendritic cells during experimental endotoxin tolerance, Acta Pharmacologica Sinica, 2009, pp. 582-588, Issue: 30, DOI: 10.1038/aps.2009.34