| Infectious disease is a large spectrum of diseases which threaten human health, and Gram-negative bacilli (GNB) are common pathogens in clinical practice. Endotoxin/ Lipopolysaccharide (LPS) is an important component of GNB, which plays a central role in the phathophysiological processes of sepsis. Severe endotxemia may lead to acute respiratory distress syndrome, multipal organ failure, and even death. There remains no specific treatment to endotoxin up till now. Endotoxin tolerance refers to a state in which less or even no reaction is seen after high dose endotoxin challenge, characteristically with low production of proinflammatory cytokines, such as tumor necrosis factor-alpha, interferon-gamma and interleukin-1. Some data show that in endotoxin rats, endotoxin initiated evelation of pulmonary tumor necrosis factor -alpha, cytokine induced neutrophil chemoattactant, and nuclear factor-kappaB translocation is alleviated, however the pulmonary expressions of anti-inflammatory cytokines remain unclear. Moreover, some researchers argue that the blunt proinflammatory cytokine production will add more possibility to following severe infection. We have found that compared to normal control rats, endotoxin tolerant rats represent a less bacteria burden, less neutrophil infiltration and less severe inflammation in lung tissue after trachea instillation of Pseudomonas Aeruginosa, suggesting that endotoxin tolerance is helpful in the defense against microorgnisms, but the pathogenis is not well known. The innate immunity is the first defence line of the body, which will react immediately once the host is irritated by pathogens. The reaction type of the innate immunity is closely related to the acquired immunity. Dendritic cells (DCs) are the most important cells in the innate immunity and play an important role in the immunity of infectious diseases. It is commonly acknowleged that the immature DCs is the most potent antigen presenting cells. Apart from the antige presenting function, DCs have function of phagocytosis and can regulate aquired immunity by secreting cytokines. In this study, endotoxin tolerace was reproduced in mice by repeatedly i.p. injection of low dose LPS, and the concentration of interleukin-10 in serum and lung tissue was detected by ELISAbefore and six hours after the i.p. injection of high dose LPS. The secretion of interleukin-10 was compared to that of interferon-gamma. DCs of both endotoxin tolerant mice and normal control mice were isolated from bone marrow and cultured. The phagocytosis of glucan, co-stimulatory factors on cell surface and the secretion of interleukin-12 were detected to evaluate the function of DCs isolated from endotoxin tolerant mice.Methods1. Clean male C57BL/10J mice, 4-6w, were randomized into endotoxin tolerance group and normal control group. LPS (Escherichia coli 055:B5) was injected intraperitoneal for four days at the dose of lmg/kg. On the fifth day, lOmg/kg of LPS was injected to induce inflammation.2. The mice of both group were sacrificed before and six hours after the injection of high dose LPS. Serum was conserved at -20°C. Lung tissue was homogenized in 0.5% triton X-100 PBS (PH 7.2) solution and kept at — 80 °C.3. ELISA was adopted to quantitiate the level of tumor necrosis factor-alpha in serum as well as interfero-gamma, interleukin-10 in both serum and lung tissue. The cytokines production of endotoxin tolerant mice was compared to that of normal control mice.4. The precursor of DCs was isolated from bone marrow. GM-CSF and interleukin-4 were used in the cell culture, and immunol beads were adopted to select DCs.5. 2X 105/ml DCs and lmg/ml FITC-glucan (4KD) were mixed at 37"C for three hours. The phagocytosis of DCs was evalutated by FACScan and the percentage of the phagocytosis cells.6. FACScan was done to detected the expression of MHC-II, CD40, CD80, and CD86 on the surface of DCs.7. The secretion of interleukin-12 by immature and mature DCs in endotoxin tolerance group and normal control group was analysised by ELISA.8. Statistics was performed by SPSS 11.5 software. Values were expressed as mean ± SD. The unpaired t test was adopted to calculate statistical differences between and within groups respectively. Differences were considered significant at the level of p< 0.05.Results1. In normal control group the level of tumor necrosis factor-alpha in serum at six hours after the injection of high dose LPS was as 2 times as that before injection, with the data being (115.76±16.85)pg/ml to (288.18±43.00)pg/ml and the P value less than 0.001. As contrast, there wasn't significant difference before and after high dose LPS challenge in endotoxin tolerant group. It was almost same before injection in two groups.2. In normal control group the level of interferon-gamma in both serum and lung tissue was dramatically increased after the injection of high dose LPS. The serum concentration was elevated from (30.34+1.85)pg/ml to (128.85 + 53.77)pg/ml, with the P value being 0.015, while the pulmonary expression from (53.64±4.85)pg/g to (220.02±21.86)pg/g, with the P value less than 0.001. As contrasted, the serum concentration of endotoxin tolerance group was slightly increased from (30.83±2.46)pg/g to (40.74±4.90)pg/g, and the level in lung tissue was not increased.3. In endotoxin tolerance group, the concentration of interleukin-10 remained elevated in both serum and lung tissue after high dose LPS challenge, with the data of the former from (28.37+6.90)pg/ml to (68.42 + 5.72)pg/ml (pO.OOl) and the latter from (40.63 +2.82)pg/g to (95.20+ 7.68)pg/g.4. The cells from mice bone marrow was cultured with the presense of GM-CSF and IL-4, and selected with immunol beads. The harvested cells showed typical morphology of DCs under microscopic. The purance was more than 95%.5. The phagocytosis percentage of glucans of DCs isolated from endotoxin tolerant mice was higher than that of normal control mice (59% vs. 34%). The result was the representative of three independent tests and every times tested 5 mice. The FACScan showed the same result (90.81 vs. 64.52).6. The FACScan analysis showed that the MHC- II and CD80 expression on DCs isolated from endotoxin tolerant mice was higher than that from normal control mice, with the data being 88.84 vs. 45.54 and 80.89 vs. 52.09 repectively.7. The secretion of interleukin-12 of both immature and mature DCs from endotoxin tolerance mice were lower than that from normal control mice, with the data being (158.18 + 46.40)pg/ml vs. (217.35 + 7.09)pg/ml (p=0.026) and (123.70 +... |
