Lactoferrin Protects against Development of Hepatitis Caused by Sensitization of Kupffer Cells by Lipopolysaccharide

BALB/c mice were intravenously injected with lipopolysaccharide (LPS) (0.05 μg/g of body weight) 7 days after being primed with zymosan. Recombinant human lactoferrin (250 μg/g of body weight), intravenously administered 1 day before the injection of LPS, significantly lessened the severity of hepatitis, as assessed by levels of serum alanine transaminase compared to those seen when casein was administered. The transient rise of serum tumor necrosis factor alpha (TNF-α) after LPS treatment was also significantly lowered by the intravenous administration of lactoferrin, suggesting that the effect of lactoferrin was due to the suppression of TNF-α production. The following results indicate that the sites of action of lactoferrin for the suppression of the development of this type of hepatitis are Kupffer cells. Gadolinium chloride, a substance known to eliminate Kupffer cells, administered 1 day before LPS, inhibited the transient rise of TNF-α and protected against the development of hepatitis. Kupffer cells isolated from mice intraperitoneally injected with recombinant human lactoferrin became refractory to LPS. The specific interaction of recombinant human lactoferrin with the Kupffer cells was shown by a binding assay, which revealed two types of binding sites on mouse Kupffer cells. Of the two dissociation constants determined in this way, the lower dissociation constant, 0.47 × 10⁻⁶ M, was within the range of the 50% effective doses for the suppression of TNF-α production. These results suggest that recombinant human lactoferrin administered to mice suppresses the production of TNF-α by Kupffer cells by directly associating with the binding sites on these cells.     

HDL3抗脂多糖诱导的人脐静脉内皮细胞损伤

目的: 研究高密度脂蛋白3(HDL3)预处理对脂多糖(LPS) 损伤的人脐静脉内皮细胞(HUVECs)的保护作用,并探讨其可能机制。方法: 以不同浓度(50、100和200 μg/L)HDL3预处理HUVECs 18 h,再加入1 mg/L LPS作用6 h。MTT法检测细胞活力,Annexin V/PI双标后流式细胞术检测细胞凋亡,荧光显微镜观察单核细胞与HUVECs黏附,ELISA法检测培养液中VCAM-1含量,细胞免疫组织化学及Western blotting法检测细胞核内NF-κB p65的水平。结果: 与对照组相比,LPS作用后HUVECs增殖活力降低,细胞凋亡及单核细胞黏附显著增加,VCAM-1和核内NF-κB p65水平增加;HDL3预处理可以恢复HUVECs增殖活力,降低细胞凋亡及单核细胞与HUVECs的黏附,减少VCAM-1分泌,并抑制NF-κB p65的核转位,且呈浓度依赖性。结论: HDL3可拮抗LPS 对HUVECs的损伤作用,其机制可能与抑制NF-κB所介导的炎症反应有关。     

高糖对内皮祖细胞功能的损害及胰岛素的影响

目的： 探讨各浓度葡萄糖及高糖条件下胰岛素对内皮祖细胞（EPCs）增殖能力、衰老程度及分泌NO能力的影响。方法: 密度梯度离心法获取大鼠骨髓单核细胞，培养扩增EPCs并进行鉴定。收集培养第4 d的细胞分别给予不同浓度的葡萄糖（5、10、20、40 mmol/L）及在高糖（40 mmol/L）条件下给予不同浓度的胰岛素（0.1、1、10、100 nmol/L）或空白培养液干预7 d，观察细胞增殖能力、衰老细胞百分率以及NO分泌功能的变化。结果: 葡萄糖可浓度依赖性地损害EPCs的增殖，促进衰老，减少EPCs的NO分泌。在高糖条件下，胰岛素干预组与对照相比可促进EPCs增殖，延缓衰老（在1 nmol/L效应达峰值），促进NO分泌（在10 nmol/L效应达峰值）。结论: 葡萄糖剂量依赖性地损害EPCs增殖和功能，而胰岛素可对抗此作用，从而对心血管系统起到一定的保护作用。     

Role of hepatocytes and Kupffer cells in age-dependent murine hepatitis caused by a phlebovirus, Punta Toro

Punta Toro virus (PTV) infection of C57BL/6 mice results in fulminant hepatic necrosis and death in 3-week-old susceptible mice, but survival with minimal hepatocellular necrosis in 8-week-old resistant mice. Susceptibility in 3-week-old mice is associated with an earlier rise of viral titers in liver and serum than that occurring in 8-week-old resistant mice. There is also an earlier and more rapid accumulation of infectious progeny in serum vs. liver after PTV infection in both age groups, suggesting that the virus may replicate in extrahepatic sites as well as the liver. PTV infection of isolated hepatocytes and Kupffer cells from 3- and 8-week-old mice demonstrates a significant age-related difference in the ability of these cells to support replication of PTV in vitro (P less than 0.05). The age-related difference in liver cell-PTV interaction appears to be an inherent difference in the liver cells themselves, since there are no age-related differences in viral adsorption, morphogenesis, cytopathic effect, or interferon action within these cells. Thus, age-related differences in PTV replication or dissemination at extrahepatic sites, and the ability of the virus to replicate in intrahepatic sites, may be additive factors in the expression of age-related susceptibility to PTV in C57BL/6 mice.     

Induced hyporesponsiveness in rat Kupffer cells is not specific for lipopolysaccharide.

The phenomenon of lipopolysaccharide (LPS)-induced hyporesponsiveness has been reported to occur in macrophage cell lines and primary cells. Hyporesponsiveness was evidenced by a diminution or lack of production of tumour necrosis factor-alpha (TNF-alpha) after sequential doses of LPS. In order to characterize the hyporesponsive state in Kupffer cells, the production of TNF-alpha was quantified after varying the concentration of a primary low dose of LPS prior to a challenge with a high, normally stimulatory dose of LPS. The kinetics of establishment of the hyporesponsive state and the effect of varying the bacterial serotype and genus of the challenge dose were determined. The specificity of the hyporesponsive state for LPS was examined. Our results demonstrate that complete hyporesponsiveness with no detectable production of TNF-alpha (< 30 pg/ml) was achieved after a primary dose > or = 10 ng/ml. Establishment of the hyporesponsive state took place within 6 hr. Induction of hyporesponsiveness was not dependent upon the serotype or genus of the challenge dose of LPS and was not specific for LPS. Complete hyporesponsiveness was induced after a primary dose (10 micrograms/ml) of the Gram-positive bacterium Corynebacterium parvum (Cp) and was evident upon challenge with 100 micrograms/ml Cp. The data indicate that the mechanisms by which LPS and Cp induce hyporesponsiveness are not identical in that a primary dose of LPS (10 ng/ml) induced only partial hyporesponsiveness upon challenge with Cp (100 micrograms/ml). These studies improve our understanding of Kupffer cell function.     

Osteopontin protects against lung injury caused by extracellular histones

Extracellular histones are present in the airways because of cell death occurring during inflammation. They promote inflammation and cause tissue damage due to their cationic nature. The anionic phosphoglycoprotein osteopontin (OPN) is expressed at high levels during airway inflammation and has been ascribed both pro- and anti-inflammatory roles. In this study, it was hypothesized that OPN may neutralize the harmful activities of extracellular histones at the airway mucosal surface. In a model of histone-induced acute lung injury, OPN^−/− mice showed increased inflammation and tissue injury, and succumbed within 24 h, whereas wild-type mice showed lower degrees of inflammation and no mortality. In lipopolysaccharide-induced acute lung injury, wild-type mice showed less inflammation and tissue injury than OPN^−/− mice. In bronchoalveolar lavage fluid from ARDS patients, high levels of OPN and also histone–OPN complexes were detected. In addition, OPN bound to histones with high affinity in vitro, resulting in less cytotoxicity and reduced formation of tissue-damaging neutrophil extracellular traps (NETs). The interaction between OPN and histones was dependent on posttranslational modification of OPN, i.e., phosphorylation. The findings demonstrate a novel role for OPN, modulating the pro-inflammatory and cytotoxic properties of free histones.     

血清对分离枯否细胞细菌脂多糖摄取过程的影响

本文应用单克隆抗细胞抗细菌多糖抗体，利用免疫荧光及激光共聚焦扫描技术，观察了分离培养的枯否细胞有无血清条件下对二种不内发子结构细菌脂多糖摄取过程的影响，无血清状态下，枯否细胞加入Ｓ型脂多糖（Ｓ－ＬＰＳ）及Ｒ型脂多糖（Ｒｄ－ＬＰＳ）培育５ｍｉｎ后，其胞闪内抗脂多糖荧光强度显著高于空白对照水平。且Ｓ－ＬＰＳ培育后胞浆内荧光强度增高幅度高于Ｒｄ－ＬＰＳ培养后的增高幅度，１０％小牛血清状态下，Ｓ－ＬＰＳ培     

Mucosal innate response stimulation induced by lipopolysaccharide protects against Bordetella pertussis colonization

Non-specific enhancement of the airways innate response has been shown to impair lung infections in several models of infection such diverse as influenza A, Streptococcus pneumoniae, and Aspergillus niger. Our aim was to evaluate whether a similar event could operate in the context of Bordetella pertussis respiratory infection, not only to enrich the knowledge of host–bacteria interaction but also to establish immunological basis for the development of new control strategies against the pathogen. Using a B. pertussis intranasal infection model and coadministration of different TLR agonists at the moment of the infection, we observed that the enhancement of innate response activation, in a TLR4-dependent way, could efficiently impair B. pertussis colonization (P < 0.001). While LPS from different microbial sources were equally effective in promoting this effect, flagellin and poly I:C coadministration, in spite of inducing expression of innate response markers TNFα, CXCL2, CXCL10 and IL6, was not effective to prevent B. pertussis colonization. Our results indicate that during the early stage of infection, specific anti-microbial mechanisms triggered by TLR4 stimulation are able to impair B. pertussis colonization. These findings could complement our current view of the role of TLR4-dependent processes that contribute to anti-pertussis immunity.     

Recombinant OspA protects dogs against infection and disease caused by Borrelia burgdorferi.

Twenty-two specific-pathogen-free beagles were vaccinated with recombinant OspA (ospA gene derived from Borrelia burgdorferi B31) alone or with adjuvant (QuilA, Montanide ISA25, or aluminum hydroxide) at 6 weeks of age. Thirteen dogs were used as nonvaccinated controls with or without adjuvant. Three dogs were kept as contact controls and received neither vaccine nor challenge. Six weeks or 6 months after the first vaccination, the vaccinated (20 of 22) and nonvaccinated dogs (13) were challenged by exposure to adult ticks (Ixodes scapularis) naturally that were infected with B. burgdorferi (tick infection rate, > or = 60%) and that were collected from Westchester County, N.Y. Protection from infection was evaluated by culture for B. burgdorferi from skin biopsies taken near the sites of tick bites. Skin biopsies were taken at monthly intervals for 3 months. B. burgdorferi was not isolated from any of the vaccinated dogs. In contrast, 12 of 13 control dogs challenged by exposure to the ticks were culture positive. The histopathology of the joint capsules 3 months after the challenge was used to evaluate protection from arthritis. Eight of 13 control dogs showed synovitis in single or multiple joints, while only 1 of the 22 vaccinated dogs had a single focus of mild inflammation in a single joint. At the time of the challenge, the vaccinated dogs had antibody to B. burgdorferi, which was demonstrable by kinetic enzyme-linked immunosorbent assay, Western blotting (immunoblotting), and a serum growth inhibition assay. The vaccinal antibody declined gradually after the challenge, especially in dogs vaccinated with OspA without adjuvants. Antibodies in the challenge control dogs were only detectable by 4 to 6 weeks after the challenge and remained at high levels until the termination of the study. Contact control dogs showed no antibody responses or histopathologic lesions and were culture negative. By Western blot analysis, antibodies to OspA first appeared in the sera of vaccinated dogs 3 weeks after the first vaccination. The absence of additional bands after the challenge suggests that infection in vaccinated dogs was blocked. Results from this study show that vaccination with recombinant OspA protected dogs against infection and disease after an experimental challenge with B. burgdorferi by exposure to ticks.     

Relaxin protects against myocardial injury caused by ischemia and reperfusion in rat heart.

Myocardial injury caused by ischemia and reperfusion comes from multiple pathogenic events, including endothelial damage, neutrophil extravasation into tissue, platelet and mast cell activation, and peroxidation of cell membrane lipids, which are followed by myocardial cell alterations resulting eventually in cell necrosis. The current study was designed to test the possible cardioprotective effect of the hormone relaxin, which has been found to cause coronary vessel dilation and to inhibit platelet and mast cell activation. Ischemia (for 30 minutes) was induced in rat hearts in vivo by ligature of the left anterior descending coronary artery; reperfusion (for 60 minutes or less if the rats died before this predetermined time) was induced by removal of the ligature. Relaxin (100 ng) was given intravenously 30 minutes before ischemia. The results obtained showed that relaxin strongly reduces 1) the extension of the myocardial areas affected by ischemia-reperfusion-induced damage, 2) ventricular arrhythmias, 3) mortality, 4) myocardial neutrophil number, 5) myeloperoxidase activity, a marker of neutrophil accumulation, 6) production of malonyldialdehyde, an end product of lipid peroxidation, 7) mast cell granule release, 8) calcium overload, and 9) morphological signs of myocardial cell injury. This study shows that relaxin can be regarded as an agent with a marked cardioprotective action against ischemia-reperfusion-induced myocardial injury.     

Acute tryptophan pretreatment protects against behavioral changes caused by cerebral ischemia

Male gerbils (Meronies ungulata) were treated with various doses of tryptophan and the changes in spontaneous motor activity determined. Tryptophan decreased behavior at a dose of 200 mg/kg. Cerebral ischemia was produced by bilateral carotid occlusion for 5 min. This duration of ischemia produced a large increase in activity at both 6 h and 24 h postischemia. Tryptophan (200 mg/kg) prevented the ischemia-induced increases in locomotor activity. These data suggest that dietary amino acids may play a role in determining the effects of ischemia.     

Scavenger receptor pathway for lipopolysaccharide binding to Kupffer and endothelial liver cells in vitro.

We have investigated the interaction of Salmonella minnesota R595 lipopolysaccharide (ReLPS) depleted of Ca2+ and Mg2+ with both Kupffer and endothelial liver cells under serum-free conditions. Specific and saturable binding levels of 125I-ReLPS were similar in both types of cells with respect to divalent cation independence, susceptibility to proteases, and concanavalin A inhibition. By using partial structures of ReLPS, it was demonstrated that acidic 3-deoxy-D-manno-octulosonic acid residues and phosphoryl groups on lipid A are of primary importance in ReLPS binding. The role of ionic interactions in LPS recognition by the cells was further confirmed by susceptibility of the binding to competitive inhibition by polyanions. Both ReLPS and ReLPS partial structures inhibited the specific cellular binding of acetylated low-density lipoprotein (Ac-LDL) by Kupffer cells and Ac-LDL- and formaldehyde-treated albumin by endothelial cells whose cellular accumulation is mediated by a different type(s) of scavenger receptor(s). In contrast, 125I-ReLPS binding to Kupffer and endothelial cells was not competed by Ac-LDL or formaldehyde-treated albumin. Our results indicate the scavenger pathway of LPS uptake by Kupffer and endothelial cells and the primary role of LPS anionic properties in this process.     

Administration of endotoxin associated with lipopolysaccharide tolerance protects mice against fungal infection

Lipopolysaccharide (LPS) pretreatment of mice resulted in a significantly enhanced survival after disseminated Cryptococcus neoformans infection. The survival was associated with reduced fungal burden in tissues. LPS-pretreated mice had lower levels of cytokines in blood, spleen, and lungs and higher levels in brain. Pentoxifylline abolished the beneficial effect of LPS pretreatment.     

GITRL对Kupffer细胞内脂多糖诱导的吲哚胺2,3双加氧酶的作用研究

目的:研究糖皮质激素诱导的TNF受体配体(GITRL)对Kupffer细胞(KCs)内脂多糖(LPS)诱导的吲哚胺2,3双加氧酶(IDO)的影响。方法:分离小鼠KCs后分为5组:Control组,只加培养基;LPS组,加入LPS(10μg/ml);LPS+Control siRNA组,转染Control siRNA后同LPS组;LPS+GITRLsiRNA组,转染GITRLsiRNA后同LPS组;LPS+Dex组,地塞米松(100μmol/L)处理后同LPS组。处理24小时后,分别采用免疫细胞化学染色、蛋白免疫印记和ELISA法检测KCs的GITRL、IDO表达及肿瘤坏死因子(TNF)-α分泌。结果:和Control组比较,LPS刺激后KCs上GITRL的表达明显上调(P<0.05),而沉默GITRL基因或者使用地塞米松能减弱其升高(P<0.05)。LPS可以诱导IDO和TNF-α在KCs上的高表达,然而GITRL基因沉默可以抑制其诱导的IDO和TNF-α的表达(P<0.05),同样地塞米松预处理也能够减弱其诱导的IDO和TNF-α的表达(P<0.05)。结论:GITRL介导了KCs内LPS诱导的IDO,地塞米松可通过下调GITRL的表达以降低LPS诱导的IDO。     

Urticaria caused by sensitization to aztreonam

A 72-year-old woman presented with generalized urticaria after receiving an injection of aztreonam. After successive in vitro and in vivo studies, allergy to aztreonam with a good tolerance of the other β-lactams was diagnosed.     

Uptake and degradation of glycogen by Kupffer cells.

Rat liver glycogen was isotopically labeled with [¹⁴C] glucose and isolated. The isolated glycogen was injected intravenously into a series of rats and its vascular clearance, uptake and degradation in liver was analyzed by means of labeling and ultrastructural techniques. Injected glycogen was quickly removed from serum with a $\text{t}\text{1}\text{2}$ of less than 15 min. Glycogen particles, identified in the electron microscope, were never seen to attach to the surface of Kupffer cells or hepatocytes. These particles appeared to be taken up by Kupffer cells by nonspecific pinocytosis “fluid endocytosis” e.g., as a solute with engulfed liquid. By 10 min the particles were present within single membrane bound vacuoles of Kupffer cells. At this early time point, the vacuoles did not seem to have fused with preexisting prelabeled secondary lysosomes containing ferritin. At later time points, glycogen particles were seen intermingled with ferritin. By 1, 2, and 4 hr, increasing numbers of vacuoles containing granular material believed to represent glycogen were observed. These vacuoles often showed extensive enlargement (“swelling”). By 24 hr, most glycogen particles had disappeared and granular material was prresent only in occasional lysosomes which no longer appeared swollen. The estimated half-life for glycogen in Kupffer cell lysosomes was in the range of 12 to 16 hr. This is considerably longer than for membrane proteins and lipids introduced into Kupffer cell lysosomes by means of heterophagy. Because of possible reutilization of isotope it was difficult to define the half-life of glycogen more exactly. It is concluded that glycogen is degraded in Kupffer cell lysosomes, although at a relative slow rate, in comparison with the capability of lysosomal hydrolases to digest proteins and lipids. This conclusion is in line with the general notion that glycogen degradation takes place in the cell sap and is not primarily associated with any particular organelle.     

Phenobarbital pretreatment protects against morphologic changes in rat bronchiolar epithelium caused by an impurity of malathion.

Oral administration (20 mg/kg) of O,O,S-trimethyl phosphorothioate (OOS) causes delayed toxicity in rats; ie, death occurs as late as 28 days after treatment. OOS-treated rats show morphologic changes in the bronchiolar epithelium of the lung; nonciliated (Clara) cells are fewer but larger 3 days after treatment. We have now found that pretreatment with the P-450-dependent mixed-function oxidase inducer, phenobarbital, protects against the morphologic changes caused by OOS. These results support the view that the lung is a target organ of the delayed toxicity caused by OOS and that OOS detoxification is mediated by P-450-dependent metabolism.     

Selective injury to rat liver Kupffer cells caused by beryllium phosphate: an explanation of reticuloendothelial blockade.

The i.v. administration of suspensions of beryllium phosphate (5-50 mumol/kg) to rats resulted in the vacuolation of hepatic Kupffer cells within 3 h. After 6 h necrotic Kupffer cells were common throughout the sinusoids of the liver but no changes were detected in the hepatic parenchymal cells during this period. A significant reduction in the numbers of intrasinusoidal cells was observed 14 h after treatment but this population had reverted to normal within 24 h. The administration of colloidal carbon to treated animals at this time did, however, demonstrate a reduction in the complement of functional endocytic cells. These results demonstrate a selective destruction of endocytic cells in the liver by this particulate toxin and the limited response by the organ to this injury. These observations are the most probable explanation for the reticuloendothelial blockade known to be caused in vivo by beryllium phosphate.     

IL-12 protects mice against pulmonary and disseminated infection caused by Cryptococcus neoformans

We examined the role of IL-12 in host resistance to Cryptococcus neoformans using a murine model of pulmonary and disseminated infection. In this model, mice were infected intratracheally with viable yeast cells. Mice untreated with IL-12 allowed an uncontrolled multiplication of yeast cells in the lung with infiltrations of few inflammatory cells, and a cryptococcal dissemination to the brain and meningitis by 3 weeks, resulting in death of all animals within 4–6 weeks. IL-12, when administered from the day of tracheal infection for 7 days, induced a marked infiltration of inflammatory cells, consisting mostly of mononuclear cells, and significantly reduced the number of viable yeast cells in the lung. The treatment suppressed brain dissemination, as shown by a marked reduction of yeast cells in the brain and prevention of meningitis. These effects resulted in a significant increase in the survival rate of infected mice. In contrast, late administration of IL-12 commencing on day 7 after instillation of yeast cells failed to protect the mice against infection with C. neoformans. In further experiments, early administration of IL-12 markedly induced interferon-gamma (IFN-γ) mRNA in the lungs of infected mice, while no IFN-γ mRNA was detected without this treatment. Our results indicate that IL-12 is effective when administered in the early period of pulmonary cryptococcal infection.     

Histamine protects T cells and natural killer cells against oxidative stress.

Oxidative stress inflicted by monocytes/macrophages (MO) is recognized as an important immunosuppressive mechanism in human neoplastic disease. We report that two types of lymphocytes of relevance for protection against malignant cells, T cells and natural killer (NK) cells, became anergic to the T cell and NK cell activator interleukin-2 (IL-2) after exposure to MO-derived reactive oxygen metabolites and subsequently acquired features characteristic of apoptosis. The MO-induced anergy and apoptosis in T cells and NK cells were reversed by histamine, an inhibitor of reactive oxygen metabolite synthesis in MO. We propose that strategies to circumvent oxidative inhibition of lymphocytes may be of benefit in immunotherapy of neoplastic disease.