Annexin-A1 gene expression during liver development and post-translation modification after experimental endotoxemia

Objective and design: We have previously reported a role for annexin-A1 in liver proliferation and tumorogenicity as well as its action as an acute phase protein in a model of endotoxemia in interleukin-6 null mice. Material and methods: In this study, we have investigated the analysis of the gene and protein expression in annexin-A1 null mice and the wild type livers during foetal and adult life, and in the presence of a proinflammatory stimulus. Results: The data indicate a link between the expression of the annexin-A1 as serine-phosphorylated-protein during early events of the inflammatory response and as tyrosine-phosphorylated-form at later time-points, during the resolution of inflammation. Conclusions: The study of annexin-A1 post-translation modification may promote a new annexin-A1 peptide discovery programme to treat specific pathologies. Received 13 June 2007; returned for revision 10 August 2007; accepted by M. Parnham 27 August 2007     

Cell microvesicles during experimental endotoxemia

The dynamics of microvesicle formation in arterial blood in generalized Schwartzman phenomenon was studied. Successive (with 24-h interval) intravenous injections of endotoxin to rabbits in a dose of 1 mg/kg and 3 mg/kg caused an increase in the content of microvesicles in the blood, some of them containing ecto-5′-nucleotidase. Biphasic changes in arterial blood clotting time and erythrocyte hemolysis were observed. __________ Translated from Byulleten’ Eksperimental’noi Biologii i Meditsiny, Vol. 142, No. 11, pp. 517–520, November, 2006     

Cytokine modulation of atopic dermatitis filaggrin skin expression

BACKGROUND: Atopic dermatitis (AD) is a chronic inflammatory skin disease that is characterized by a defective skin barrier function. Recent studies have reported mutations of the skin barrier gene encoding filaggrin in a subset of patients with AD. OBJECTIVE: We investigated whether reduced filaggrin expression was found in patients with AD who were not carriers of known filaggrin mutations and whether filaggrin expression was modulated by the atopic inflammatory response. METHODS: Filaggrin expression was measured in skin biopsies and cultured keratinocytes using real-time RT-PCR and immunohistochemistry. Filaggrin loss-of-function mutations were screened in a total of 69 subjects. RESULTS: Compared with normal skin, filaggrin expression was significantly reduced (P < .05) in acute AD skin, with further reduction seen in acute lesions from 3 European American subjects with AD who were heterozygous for the 2282del4 mutation. This was confirmed by using immunohistochemistry. AD skin is characterized by the overexpression of IL-4 and IL-13. Keratinocytes differentiated in the presence of IL-4 and IL-13 exhibited significantly reduced filaggrin gene expression (0.04 +/- 0.01 ng filaggrin/ng glyceraldehyde 3-phosphate dehydrogenase; P < .05) compared with media alone (0.16 +/- 0.03). CONCLUSION: Patients with AD have an acquired defect in filaggrin expression that can be modulated by the atopic inflammatory response. CLINICAL IMPLICATIONS: The atopic immune response contributes to the skin barrier defect in AD; therefore, neutralization of IL-4 and IL-13 could improve skin barrier integrity.     

磷脂酰肌醇特异性磷脂酶C对内毒素血症大鼠肝组织CD14表达的抑制作用

目的观察磷脂酰肌醇特异性磷脂酶C(PI—PLC)对大鼠内毒素血症时肝组织中Kuiter细胞(KCs)的活性、CD14 mRNA的表达和血浆细胞因子释放的作用。方法将Wistar大鼠90只,随机分为LPS组(静注LPS 5mg／kg)、PI—PLC组(注LPS前30min注PI-PLC 100U／kg)和生理盐水NS组。分别于注射前及注射后1、3、6和12h取材,每组每时相点6只,测定血浆内毒素(鲎试剂基质偶氮显色法)、LBP及TNF-α(均用ELISA法)和IL-6(放免法)的含量,用RT-PCR检测肝组织中CD14 mRNA的表达,并观察其形态学变化。结果LPS组LBP、TNF-α、IL-6的含量和CD14mRNA的表达明显增加,并伴有KCs激活,数量增多,体积增大,吞噬功能增强,肝细胞出现变性和坏死等;而PI-PLC处理组所致的上述变化明显减轻。结论PI—PLC对内毒素所致肝组织内KCs的激活有一定抑制作用。     

Cytokine expression in the liver during the early phase of murine tularemia.

Cytokine expression was determined in the livers of mice inoculated subcutaneously with Francisella tularensis LVS. During the first 48 h of infection, there was a logarithmic increase of bacteria in the liver, with a doubling time of 2.5 h. Within 48 h, tumor necrosis factor alpha (TNF-alpha), interleukin 10 (IL-10), IL-12, and gamma interferon (IFN-gamma) mRNAs were expressed, and production of TNF-alpha and IFN-gamma was demonstrated. There was no expression within 96 h of mRNA from IL-2, IL-3, or IL-4. After subcutaneous inoculation of heat-killed LVS, no expression of any of the cytokine mRNAs and no increase in the levels of TNF-alpha or IFN-gamma occurred. The expression of TNF-alpha, IL-12, and IFN-gamma is held to be important to evoke an early T-cell-independent host defense against F. tularensis as well as to drive the expansion of a protective Th1 cell response.     

Up-regulated expression of HGF in rat liver cells after experimental endotoxemia: a potential pathway for enhancement of liver regeneration

A lipopolysaccharide (LPS)-induced inflammation prior to an hepatic resection has been shown to enhance liver regeneration in rat. The aim of the present study was to investigate the expression of hepatocyte growth factor (HGF) and its c-Met receptor under such experimental conditions. Animals were submitted to a two-third hepatectomy or a LPS challenge carried out 12 h prior to resection. Non parenchymal and parenchymal cells were isolated from livers obtained at various times post-hepatectomy. Quantitative RT-PCR for HGF and c-Met mRNAs were performed from total liver or purified cell fractions and HGF mRNA was also analyzed by in situ RT-PCR on liver sections. A LPS challenge alone induced a marked up-regulation of HGF mRNA level in whole liver and isolated hepatocytes. Furthermore, when partial hepatectomy (PH) was preceded by a LPS challenge, an increase of HGF mRNA level was seen in whole liver and contrasted with a decreased level in non parenchymal cells. These results were confirmed by in situ RT-PCR. In isolated hepatocytes from endotoxemic rats, the mRNA level for the LPS-specific membranous receptor mCD14 was markedly up-regulated and even more so when LPS was followed by PH. Moreover, a TNFalpha challenge alone induced an up-regulation of HGF mRNA in hepatocytes and a down-regulation in non parenchymal cells (NPCs). Overall, when a LPS challenge is given prior to PH the major source of hepatic HGF appears to be the hepatocyte itself rather than NPCs. An autocrine HGF/c-Met loop which promotes the proliferative potential of the hepatic parenchymal cell and participates in liver regeneration is postulated.     

Phenotypic modulation in lipocytes in experimental liver fibrosis

The presence of a-smooth muscle actin (smA)-positive cells has recently been reported in the fibrotic liver. Lipocytes have been considered to play important roles in hepatic fibrosis. However, the relation of the a-smA-positive cells and lipocytes has not been determined. The biological implication of a-smA expression remains unknown. To study these questions, we carried out double immunofluorescent staining of a-smA and desmin (a marker for lipocytes), or a-smA and collagen, and double immunohistochemical staining of a-smA and 5-bromo-2'-deoxyuridine (BrdUrd) in carbon tetrachloride-induced fibrotic rat livers. In normal and control livers, a-smA-positive cells were not seen in the lobules, whereas scattered desmin-positive cells were present. With the development of hepatic fibrosis, a-smA was expressed only in a portion of desmin-positive cells located predominantly around collagen bundles. A number of a-smA-positive cells in the lobules were labelled with BrdUrd. These results suggest phenotypic modulation in lipocytes and differentiation of lipocytes towards myofibroblast-like cells, since a-smA is expressed with desmin in myofibroblasts in scar tissue. The expression of a-smA may be related to events of the fibrotic process, such as tissue contraction or fibrogenesis per se.     

An immunocytochemical and in situ hybridization analysis of annexin 1 expression in rat mast cells: modulation by inflammation and dexamethasone

The presence and localization of the anti-inflammatory protein annexin 1 (also known as lipocortin 1) in perivenular rat mast cells was investigated here. Using the rat mesenteric microvascular bed and a combination of morphologic techniques ranging from immunofluorescence to electron microscopy analyses, we detected the presence of annexin 1 in discrete intracellular sites, both in the nucleus and in the cytoplasm. In resting mast cells, most of the protein pool (approximately 80% of the cytosolic portion) was localized to cytoplasmic granules. In agreement with other cell types, treatment of rats with dexamethasone (0.2 mg/kg, ip) increased annexin 1 expression in mast cells, inducing a remarkable appearance of clusters of protein immunoreactivity. This effect was most likely the result of de novo protein synthesis as determined by an increase in mRNA seen by in situ hybridization. Triggering an ongoing experimental inflammatory response (0.3 mg of carrageenin, ip) increased annexin 1 mRNA and protein levels. In conclusion, we report for the first time the localization of annexin 1 in connective tissue mast cells, and its susceptibility not only to glucocorticoid hormone treatment, but also to an experimental acute inflammatory response.     

Cytokine production during experimental infection of mice with tick-borne encephalitis virus

An experiment with BALB/c mice, infected with a lethal dosage of the virus of tick-borne encephalitis (TE), strain 205, was accompanied by pronounced growing concentrations of the IL-1 beta, IL-6, IL-10 and TNF alpha cytokines in the blood serum of animals. The maximum values of the above cytokines were determined at the infection terminal stage. A reliably less pronounced growth of concentrations of IL-1 beta, TNF alpha and IL-10 was found in animals infected with a non-lethal TE dosage. The concentration of IL-6 in the blood serum of animals, infected with a non-lethal dosage of the virus, changed during the whole observation period. The dynamics of cytokines in the blood serum of mice, infected with a lethal dosage of the TE virus, suggests the development of SIRS at the infection terminal stage.     

诱生型一氧化氮合酶抑制剂硫酸甲基异硫脲对内毒素血症大鼠肠系膜淋巴管一氧化氮合酶表达的影响

目的 :研究内毒素血症中诱生型一氧化氮合酶 (induciblenitricoxidesyhthases,iNOS)抑制剂硫酸甲基异硫脲(S Methy1isothioureasulfate,SMT)对大鼠肠系膜淋巴管一氧化氮合酶 (nitricoxidesyhthase,NOS)表达的影响。探讨在内毒素血症中一氧化氮 (nitricoxide,NO)对肠系膜淋巴微循环的影响。方法 :利用NADPH 黄递酶组织化学染色方法观察NOS在大鼠肠系膜淋巴管的表达 ,用硝酸还原酶法测定血清中NO的含量。结果 :脂多糖(lipopolysaccharide,LPS)组大鼠随时间延长肠系膜淋巴管染色强度明显加深 ,血清NO含量显著升高。LPS SMT组染色强度比LPS组降低 ,血清NO含量显著下降。结论 :在内毒素血症中 ,SMT可能通过抑制NOS的活性而起保护作用。     

Fe allocation in liver during early stages of endotoxemia in Fe-overload rats

The hypothesis of this study was that alterations in Fe distribution triggered by lipopolysaccharide (LPS) administration were affected in vivo by Fe overload. Lipopolysaccharide treatment by itself significantly decreased Fe content in serum and increased the blood NO-hemoglobin (NO-Hb) EPR signal and nitrotyrosine protein content in liver, as compared to values in control animals. Fe overload (produced by Fe-dextran ip administration) caused an increase, as compared to values in control animals, in Fe content in serum, and a significant enhancement in ferritin (Ft) content, Fe content in Ft, the labile Fe pool (LIP), and the protein carbonyl content in the liver. The simultaneous administration of LPS and Fe-dextran lead to a significant increase in the Fe content in serum, blood NO-Hb EPR signal, the content of Fe, Fe in Ft, LIP, protein carbonyl, and nitrotyrosine protein in liver, as compared to values in control animals. The data reported here indicate that the protective strategy against endotoxemia of sequestering serum Fe content is not fully operative under Fe overload conditions. However, the oxidative condition of the liver does not seem to be being affected, since endogenous mechanisms were able to regulate the amount of catalytically active Fe to the same levels observed after Fe-dextran administration, even in the presence of LPS, over the initial six-hour period.     

Neutrophil interaction with inflamed postcapillary venule endothelium alters annexin 1 expression

Annexin 1 (ANX-A1) exerts antimigratory actions in several models of acute and chronic inflammation. This is related to its ability to mimic the effect of endogenous ANX-A1 that is externalized on neutrophil adhesion to the postcapillary endothelium. In the present study we monitored ANX-A1 expression and localization in intravascular and emigrated neutrophils, using a classical model of rat peritonitis. For this purpose, a pair of antibodies raised against the ANX-A1 N-terminus (ie, able to recognize intact ANX-A1) or the whole protein (ie, able to interact with all ANX-A1 isoforms) was used by immunofluorescence and immunocytochemistry analyses. The majority ( approximately 50%) of ANX-A1 on the plasma membrane of intravascular neutrophils was intact. Extravasation into the subendothelial matrix caused loss of this pool of intact protein (to approximately 6%), concomitant with an increase in total amount of the protein; only approximately 25% of the total protein was now recognized by the antibody raised against the N-terminus (ie, it was intact). In the cytoplasm of these cells, ANX-A1 was predominantly associated with large vacuoles, possibly endosomes. In situ hybridization confirmed de novo synthesis of ANX-A1 in the extravasated cells. In conclusion, biochemical pathways leading to the externalization, proteolysis, and synthesis of ANX-A1 are activated during the process of neutrophil extravasation.     

Time-dependent expression of annexin 1 in a model of chronic granulomatous inflammation

OBJECTIVE AND DESIGN: To determine the expression pattern and distribution of the glucocorticoid-inducible protein annexin 1 (ANXA1) in a murine model of chronic granulomatous inflammation. MATERIALS OR SUBJECTS: TO Mouse. TREATMENT: Chronic granulomatous inflammation was induced by injecting into dorsal sub-cutaneous air-pouches in mice, a mixture of croton oil and Freund's complete adjuvant (CO/FCA). METHODS: Western and northern analysis, corticosterone assay, and immunohistochemistry. Statistical analysis was performed using ANOVA followed by Tukey's pair-wise comparisons or Dunnett's multiple comparisons. RESULTS: ANXA1 protein levels changed significantly throughout the 4-week time course, with an initial peak at day 7 and a later elevation at 28 days. ANXA1 mRNA levels peaked at days 1 and 3, with a significant decline at day 7 followed by an upward trend to day 28. Plasma corticosterone measurements taken throughout the time course revealed an increase from 14 days onward, suggesting that corticosterone does not influence ANXA1 expression during the initial stages of the model. Immunogold staining revealed that ANXA1 expression in the inflamed tissue was mainly in extravasated neutrophils, with intact protein (37 kDa) being predominantly observed on the cell membrane. CONCLUSIONS: The pattern of ANXA1 expression indicates that infiltrated neutrophils are responsible for the majority of ANXA1 present both at early and later stages of this model of granulomatous inflammation.     

Phospholipase A2 activity and lipid peroxidation during endotoxemia under conditions of experimental peritonitis

The severity of endotoxemia in peritonitis depends on morphofunctional state of the intestine determined by the intensity of free-radical lipid peroxidation and phospholipase A₂ activity, which are the highest on day 1 postoperation. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 129, No. 1, pp. 31–33, January, 2000     

Regulation of TREM expression in hepatic macrophages and endothelial cells during acute endotoxemia

Triggering receptor expressed on myeloid cells (TREM) regulates inflammatory responses to lipopolysaccharide (LPS). In these studies, we analyzed the expression of TREM in hepatic macrophages and endothelial cells which play a central role in LPS clearance. LPS administration to C3H/HeOuJ mice resulted in a rapid induction of TREM-1 and TREM-3, but a decrease in TREM-2 in liver macrophages and endothelial cells. The observation that TREM family members are detectable in endothelial cells is novel and demonstrates that their expression is not limited to myeloid cells. LPS-induced alterations in TREM expression were not evident in cells from C3H/HeJ TLR-4 mutant mice, indicating that the response is dependent on TLR-4. IL-1beta and TNFalpha upregulated TREM-1 and TREM-3 expression and suppressed TREM-2 expression in macrophages and endothelial cells. This activity involved PI3-kinase and p38 MAP kinase signaling. Interestingly, no significant differences were noted in TREM expression between wild-type and TNFR1-/- mice treated with LPS. Treatment of macrophages and endothelial cells with LPS upregulated expression of nitric oxide synthase-2 (NOS-2). This was blocked by TREM-1 Fc/fusion protein, indicating that TREM-1 mediates LPS-induced NOS-2 expression. These results suggest that TREM proteins are important in the inflammatory response of hepatic macrophages and endothelial cells to acute endotoxemia.     

Cytokine therapy of experimental purulent wounds

The effect of naturally occurring cytokine complex on aseptic and purulent wounds is examined in guinea pigs. Healing of aseptic wounds treated with the complex is 1.4 times faster than in the control: the rate of wound contraction reaches the maximum after 5–6 daysvs. 10–11 days in the control. Healing of these wounds occurs without pronounced inflammatory processes. Healing, epithelization, and wound cleansing are significantly faster in cytokine treated wounds than in the control. Histological analysis reveals formation of delicate cicatrices in cytokine-treated wounds, while rough cicatrices are observed in the control. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 123, No. 6, pp. 680–683, June, 1997     

Superoxide anion generation in the liver during the early stage of endotoxemia in rats

Infiltration of polymorphonuclear neutrophils (PMN) in the rat liver 3 hr after an intravenous (IV) injection of a sublethal dose of Escherichia coli lipopolysaccharide (LPS) was observed without any significant alteration in the total number of Kupffer and endothelial cells. Since previous studies have demonstrated that phagocytic cells in the liver were in a state of metabolic activation under similar experimental conditions, we investigated the in vitro generation of superoxide anion (O2-) by this cell type following the administration of LPS. Kupffer cells from normal rats did not release O2-, in contrast to those obtained from LPS-treated rats. The generation of O2- by Kupffer cells from endotoxic rats was elevated from 3.0 +/- 1.9 nmol/10(6) cells/60 min (mean +/- SD) in the absence of macrophage (M phi) activators, to 5.0 +/- 2.36, 11.33 +/- 5.40, and 4.33 +/- 0.90 in the presence of opsonized zymosan, phorbol myristate acetate (PMA), and the calcium ionophore A23187, respectively. Hepatocytes from normal or endotoxic rats did not produce detectable O2-. Endothelial cells from LPS-treated rats generated less than 0.8 nmol/10(6) cells in the presence of zymosan. PMN that accumulated in the livers of endotoxic rats released O2- only in the presence of zymosan (8.12 +/- 5.40), PMA (15.43 +/- 5.84), or A23187 (1.70 +/- 0.12). The O2- generation by blood monocytes and PMN increased significantly after endotoxin administration and in the presence of activators. These results suggest that the hypermetabolic state of phagocytic cells in the liver shortly after LPS treatment may be correlated with the increased generation of O2-. The latter may subsequently contribute to the induction of hepatic injury in endotoxemia.