Glial cell activity is maintained during prolonged inflammatory challenge in rats

We evaluated the expression of glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), ionized calcium binding adaptor protein-1 (Iba-1), and ferritin in rats after single or repeated lipopolysaccharide (LPS) treatment, which is known to induce endotoxin tolerance and glial activation. Male Wistar rats (200-250 g) received ip injections of LPS (100 µg/kg) or saline for 6 days: 6 saline (N = 5), 5 saline + 1 LPS (N = 6) and 6 LPS (N = 6). After the sixth injection, the rats were perfused and the brains were collected for immunohistochemistry. After a single LPS dose, the number of GFAP-positive cells increased in the hypothalamic arcuate nucleus (ARC; 1 LPS: 35.6 ± 1.4 vs control: 23.1 ± 2.5) and hippocampus (1 LPS: 165.0 ± 3.0 vs control: 137.5 ± 2.5), and interestingly, 6 LPS injections further increased GFAP expression in these regions (ARC = 52.5 ± 4.3; hippocampus = 182.2 ± 4.1). We found a higher GS expression only in the hippocampus of the 6 LPS injections group (56.6 ± 0.8 vs 46.7 ± 1.9). Ferritin-positive cells increased similarly in the hippocampus of rats treated with a single (49.2 ± 1.7 vs 28.1 ± 1.9) or repeated (47.6 ± 1.1 vs 28.1 ± 1.9) LPS dose. Single LPS enhanced Iba-1 in the paraventricular nucleus (PVN: 92.8 ± 4.1 vs 65.2 ± 2.2) and hippocampus (99.4 ± 4.4 vs 73.8 ± 2.1), but had no effect in the retrochiasmatic nucleus (RCA) and ARC. Interestingly, 6 LPS increased the Iba-1 expression in these hypothalamic and hippocampal regions (RCA: 57.8 ± 4.6 vs 36.6 ± 2.2; ARC: 62.4 ± 6.0 vs 37.0 ± 2.2; PVN: 100.7 ± 4.4 vs 65.2 ± 2.2; hippocampus: 123.0 ± 3.8 vs 73.8 ± 2.1). The results suggest that repeated LPS treatment stimulates the expression of glial activation markers, protecting neuronal activity during prolonged inflammatory challenges.

Human monocytes tolerant to LPS retain the ability to phagocytose bacteria and generate reactive oxygen species

Tolerance to lipopolysaccharide (LPS) occurs when animals or cells exposed to LPS become hyporesponsive to a subsequent challenge with LPS. This mechanism is believed to be involved in the down-regulation of cellular responses observed in septic patients. The aim of this investigation was to evaluate LPS-induced monocyte tolerance of healthy volunteers using whole blood. The detection of intracellular IL-6, bacterial phagocytosis and reactive oxygen species (ROS) was determined by flow cytometry, using anti-IL-6-PE, heat-killed Staphylococcus aureus stained with propidium iodide and 2',7'-dichlorofluorescein diacetate, respectively. Monocytes were gated in whole blood by combining FSC and SSC parameters and CD14-positive staining. The exposure to increasing LPS concentrations resulted in lower intracellular concentration of IL-6 in monocytes after challenge. A similar effect was observed with challenge with MALP-2 (a Toll-like receptor (TLR)2/6 agonist) and killed Pseudomonas aeruginosa and S. aureus, but not with flagellin (a TLR5 agonist). LPS conditioning with 15 ng/mL resulted in a 40 percent reduction of IL-6 in monocytes. In contrast, phagocytosis of P. aeruginosa and S. aureus and induced ROS generation were preserved or increased in tolerant cells. The phenomenon of tolerance involves a complex regulation in which the production of IL-6 was diminished, whereas the bacterial phagocytosis and production of ROS was preserved. Decreased production of proinflammatory cytokines and preserved or increased production of ROS may be an adaptation to control the deleterious effects of inflammation while preserving antimicrobial activity.

Inhibition of IKKα binding to IL-1β promoter by p50 in LPS tolerant THP-1 cells / 中华微生物学和免疫学杂志

Objective To study the effect of p50 on IKKα at IL-1β promoter in LPS tolerant cells and to reveal the mechanism of the inhibition of IL-1β mRNA by pS0. Methods THP-1 human promono-cyte model of endotoxin tolerance that simulates the sepsis leukocyte phenotype was used. Chromatin immu-noprecipitation assay(CHIP) and real-time PCR were applied to quantify the binding of p50 and IKKα to IL-1βpromoter. IL-1β mRNA transcription was studied after knocking-down of p50 and/or IKKα. Results With LPS stimulation, p50 binding did not reduce but somewhat increased at IL-1β promoter in tolerant THP-1 cells. Knocking-down of p50 increased the transcription of IL-1β mRNA, which revealed the inhibi-tory effect of p50 in tolerant cells. In contrast, the accumulation of IKKα to IL-1β promoter decreased with LPS stimulation in tolerant cells; However, IKKα binding increased after p50 gene knock-down. In the meantime, IL-1β mRNA transcription increased; At last, IL-1β mRNA decreased again after double-knoc-king down of p50 and IKKα. Conclusion p50 is an inhibitory protein at IL-1β promoter in tolerant THP-1 cells. The unresponsiveness of IL-1β mRNA transcription to LPS at least partly results from the inhibition of IKKα binding to IL-1β promoter by p50.

Mechanisms of Lipoplysaccharide-induced Lipopolysaccharide Tolerance in the Expression of TNF-alpha and IL-8 in Peripheral Blood Monocytes / 결핵

BACKGROUND: Monocytes/macrophages play a central role in determining the host response during Gram-negative infection through secretion of a variety of mediators after stimulation of LPS. Even though cytokine production has been shown to play an important role in host defense during sepsis cytokine release may also lead to tissue injury. Thus, regulation of macrophage response to LPS is critical for host survival during Gram-neg-alive sepsis. In animals exposed to nonlethal doses of endotoxin a characteristic hyporesponsiveness to subsequent administration of endotoxin has been observed. This phenomenon was knowm as 'LPS tolerance'. However, little information is availavble regarding the underlying mechanism of U)S tolerance. METHOD: Peripheral blood monocyte(PBMC) was isolated from peripheral blood of normal volunteers by adhesion purification method. To evaluate conditions to obtain LPS tolerance. preculture was carried out with LPS at 10ng/ml for 24 hours. For stimulation culture plates were washed two times and were stimulated with LPS at 1ng/ml for 4, 6 and 26 hours. To assess the underlying mechanisms of LPS tolerance, autologous serum, PMA, anti-CD14 Ab, Indomethacin or PGF2 were added to preculture solution respectively. Cytokine concentrations in culture supernatants were measured using ELISA for TNF-α and IL-8 and mRNA of TNF-α and IL-8 were determined by Northern blot analysis. RESULTS: The exposure of PBMC to low dose of LPS suppressed the cytokine production and mRNA expression of TNF-α, but not IL-8. Anti-CDl4 Ab partially recovered production of TNF-α which was suppressed by preculture with low dose LPS. The preculture with PMA induces US tolerance, as preculture with low dose LPS. CONCLUSION: LPS tolerance to TNF-α is regulated pretranslationally and is influenced by protein kinase C pathway and CD14.