IL‐10 deficiency blocks the ability of LPS to regulate expression of tolerance‐related molecules on dendritic cells

Abstract

Interleukin-10 (IL-10) is an anti-inflammatory cytokine that plays an important role in regulating the local inflammatory immune response, but regulatory mechanisms of this cytokine have not been fully elucidated. Here, we demonstrate that IL-10 deficiency renders LPS treatment ineffective in regulating the expression of CD40, CD80, CD86, B7-H2, and B7-DC on dendritic cells (DCs) and blocks upregulation of IL-27. This inability to respond to LPS was found in both IL-10−/− bone marrow derived and splenic DCs. Compared with wild-type DCs, IL-10−/− DCs expressed similar levels of TLR4 and CD14, but produced less LPS-binding protein. The deficiency in LPS-binding protein production may explain the failure of IL-10−/− DCs to respond normally to LPS.Moreover, lack of IL-10 modulated the proportions of CD11c+CD8+ and CD11c+B220+ DCs, which play an important role in local inflammatory responses and tolerance. IL-10 deficiency also blocked expression of galectin-1, CD205, and CD103, which are necessary for central and peripheral tolerance. While they did not respond to LPS, IL-10−/− DCs produced increased levels of IL-6 and CCL4 after TNF-α treatment. Together, our results demonstrate that IL-10 deficiency affects the immune functions of DCs, which may contribute to the increased severity of autoimmune diseases seen in IL-10−/− mice.

Immunotherapy using lipopolysaccharide-stimulated bone marrow-derived dendritic cells to treat experimental autoimmune encephalomyelitis

“…or standard error of the arithmetic mean (s.e.m.). Results were regarded as showing a significant difference if the P-value was less than 0·05 [18][19][20].…”

“…As described previously [18,19], femurs and tibiae were isolated from muscle tissue of mice. The intact bones were then sterilized with 70% ethanol for 5 min and washed with phosphate-buffered saline (PBS).…”

“…As described previously [18,19], leucocytes from bone marrow were fed in bacteriological 100-mm Petri dishes (Falcon, Becton Dickinson, Heidelberg, Germany) at 2 × 10 6 cells per dish. Cells were cultured in RPMI-1640 complete medium (Gibco-BRL, Eggenstein, Germany), including penicillin (100 U/ml; Sigma, St Louis, MO, USA), streptomycin (100 U/ml; Sigma), L-glutamine (2 mM; Sigma), 2-mercaptoethanol (2-ME, 50 μM; Sigma), 10% heatinactivated and filtered (0·22 μm; Millipore, Inc., Bedford, MA, USA) fetal calf serum (FCS; Sigma) and granulocytemacrophage colony-stimulating factor (GM-CSF; PeproTech, Rocky Hill, NJ, USA) at 20 ng/ml at day 0 (10 ml medium per dish).…”

“…Cells were washed twice with 5% FCS in PBS at 300 g for 5 min, fixed with 5% formalin in PBS at 4°C for 2 h and then permeated for intracellular staining [18][19][20].…”

“…Assays were conducted according to the manufacturer's instructions. Plates were read out in Labsystems Multiskan MCC/340 (Fisher Scientific, Suwanee, GA, USA) and data were analysed using DSJV ELISA software (Fisher Scientific) [18][19][20].…”