Excretory-secretory products from Fasciola hepatica induce eosinophil apoptosis by a caspase-dependent mechanism

Eosinophils (Eo) are known to be important effector cells in the host defense against helminth parasites. Excretory-secretory products (ESP) released by helminths have shown wide immunomodulatory properties, such as the induction of cellular apoptosis. We investigated the ability of ESP from Fasciola hepatica to induce Eo apoptosis. In this work, we observed that ESP induced an early apoptosis of rat peritoneal eosinophils and that this phenomenon was time- and concentration-dependent. Furthermore, we demonstrated that activation of protein tyrosine kinases (TyrK) and caspases were necessary to mediate the Eo apoptosis induced by the ESP, and that carbohydrate components present in these antigens were involved in this effect. We have described for the first time the ability of ESP from F. hepatica to modify the viability of Eo by apoptosis induction. Besides that, we have observed Eo apoptosis in the liver of rats 21 days after F. hepatica infection. The diminution in Eo survival in early infection could be a parasite strategy in order to prevent a host immune response.     

A20 promotes Brucella intracellular growth via inhibition of macrophage cell death and activation

The zinc-finger protein A20 has crucial physiological functions as a dual inhibitor of macrophage activation and apoptosis in tumor necrosis factor receptor1 (TNFR1) signaling pathway. Brucella infection can induce A20 expression in macrophages. Here, we hypothesize that A20 promotes Brucella intracellular growth via inhibition of activation and apoptosis of macrophages. To test this hypothesis, we stably incorporated mouse A20-shRNA into the RAW264.7 cells by lentiviral gene transfer to successfully knockdown A20. A20-deficient RAW264.7 cells were subsequently challenged with Brucella abortus and colony formation units (CFUs) of bacteria, TNFα production, NF-kB activation, macrophages apoptosis and cell death were evaluated. The A20 knockdown was shown to effectively promote B. abortus-stimulated TNFα release, NF-kB activation and macrophage cell death, which suppressed B. abortus intracellular replication. Unexpectedly, deficiency of A20 failed to lead to B. abortus-induced macrophage apoptosis. A20 deficiency coupled NF-kB inhibition promoted caspase-8 dependent B. abortus-induced macrophage apoptosis. These findings provide a novel mechanism by which Brucella intracellular growth within macrophages occurs through up-regulation of A20 thereby limiting activation and macrophages cell death.     

A "possible" involvement of TNF-alpha in apoptosis induction in peripheral blood lymphocytes of cats with feline infectious peritonitis

Feline infectious peritonitis (FIP) cats show a decrease in peripheral blood lymphocyte counts, and a particularly marked decrease in T cells including CD4+ and CD8+ cells. In this study, we showed that lymphopenia observed in FIP cats was due to apoptosis, and that the ascitic fluid, plasma, and culture supernatant of peritoneal exudate cells (adherent cells with macrophage morphology, or PEC) from FIP cats readily induced apoptosis in specific pathogen-free cat peripheral blood mononuclear cells, particularly CD8+ cells. In addition, TNF-alpha released from macrophages and TNF-receptor (TNFR) 1 and TNFR2 mRNA expression in lymphocytes were closely involved in this apoptosis induction. In particular, in CD8+ cells cultured in the presence of the PEC culture supernatant, the expression levels of TNFR1 and TNFR2 mRNA were increased, indicating that CD8+ cells are more susceptible to apoptosis induction by TNF-alpha than other lymphocyte subsets, particularly B cells (CD21+ cells). The results of this study suggest that TNF-alpha, produced by virus-infected macrophages, is responsible for induction of apoptosis in uninfected T cells, primarily CD8+ T cells.     

牛磺鹅去氧胆酸对小鼠不同免疫细胞凋亡的影响

采用二苯胺法检测清洁级昆明小鼠给予不同剂量牛磺鹅去氧胆酸后不同免疫细胞的凋亡百分率.结果显示,低剂量牛磺鹅去氧胆酸能够显著抑制正常小鼠腹腔巨噬细胞、脾脏和血液淋巴细胞的凋亡,但对胸腺细胞凋亡无显著影响;高剂量牛磺鹅去氧胆酸能够显著抑制正常小鼠胸腺细胞和血液淋巴细胞的凋亡,对脾脏淋巴细胞凋亡无显著影响,但能够显著促进正常小鼠腹腔巨噬细胞的凋亡.由此证明牛磺鹅去氧胆酸对不同的免疫细胞的凋亡具有不同的影响,显示出其免疫调节作用.     

Mechanism of macrophage activation by chitin derivatives

In order to analyze the detailed mechanisms responsible for macrophage activation by chitin derivatives, resident peritoneal macrophages were prepared and stimulated with chitin, chitosan and low-molecular weight chitosan. Our findings were as follows: (i) chitosan induced apoptosis of peritoneal macrophages, but this did not occur when chitin or water soluble low-molecular weight chitosan were used; (ii) chitosan treatment induced activation markers, such as the major histocompatibility complex (MHC) class I, class II, Fc receptors, transferrin receptor, mannose receptor, Fas, and macrophage inflammatory protein (MIP)-2, whereas chitin and low molecular weight soluble chitosan induced only the expression of MHC class I and II molecules; (iii) apoptosis induced by chitosan was mediated by the Fas signaling pathway, in response to phagocytosis via the mannose receptor. We conclude that since chitosan activates macrophages, this may be the mechanism by which it accelerates wound healing.     

Modulation of sheep lymphocyte responses by Fasciola hepatica excretory-secretory products

The effect of Fasciola hepatica excretory-secretory products (FhESPs) on mitogen-induced proliferation of sheep peripheral blood mononuclear cells (PBMCs) and PBMC subsets (CD2(+), CD4(+), CD8(+), gammadeltaTCR(+) or CD21(+) cells) were studied. PBMCs were incubated with Concanavalin A (ConA) or phytohemagglutinin (PHA) at optimal (1 microg per well) or suboptimal (0.25 microg per well) doses and with FhESPs at several doses (1.25-20 microg per well). PBMC subsets were incubated with ConA at a suboptimal dose and with FhESPs at 5 microg per well. These cells were incubated with or without monocytes (CD14(+) cell). FhESPs slightly increased the proliferation of PBMCs stimulated with optimal doses of PHA. FhESPs (10 and 20 microg per well) inhibited the PBMCs stimulated with optimal doses of ConA. FhESP dose-dependent inhibition was observed on PBMCs stimulated with suboptimal doses of ConA. CD21(+) lymphocytes (B lymphocytes), CD14(+) cells (monocytes) and gammadeltaTCR(+) cells were not stimulated by ConA. T lymphocyte subsets (CD2(+), CD4(+) or CD8(+) cells) proliferation was decreased by FhESPs at 5 microg per well. FhESPs inhibits the ConA-induced stimulation of sheep PBMCs and sheep T lymphocyte subsets. Further studies should be done to investigate the mechanism of this FhESP immunomodulatory effect.     

硒对树突状细胞和巨噬细胞功能的调控作用

旨在探讨硒对树突状细胞（dendritic cells,DCs）和巨噬细胞功能的影响,试验将硒分别与髓源性树突状细胞（bone marrow derived dendritic cells,BMDCs）和腹腔巨噬细胞共同培养后,用流式细胞术检测未成熟BMDCs和巨噬细胞的吞噬活性以及BMDCs上的MHCⅡ、CD86、CD80和CD40的表达量,测定经硒处理后的成熟BMDCs对刺激同种异体淋巴细胞增殖和抗原递呈能力,并用ELISA检测BMDCs和巨噬细胞上清液中细胞因子（IL-12、IL-1β、IFN-γ、IL-6、IL-10、TNF-α、NO）水平的变化。结果显示,当硒的质量浓度在0.18~0.09 mg·L^-1时,BMDCs和巨噬细胞的吞噬活性显著增强（P<0.05）,并且BMDCs上的MHCⅡ、CD86和CD80的表达量显著升高（P<0.05）,对刺激同种异体淋巴细胞的增殖和抗原递呈能力也显著增强（P<0.05）。此外,在BMDCs的上清中,IFN-γ、IL-12和IL-10的含量显著升高（P<0.05）;在巨噬细胞的上清中,IFN-γ、TNF-α和NO的含量显著升高（P<0.05）。结果表明,一定质量浓度的硒可以增强树突状细胞和腹腔巨噬细胞的功能,值得进一步探究硒对免疫功能的影响。     

Association of parainfluenza-3 virus with bovine macrophages and blood cells: an in vitro study

Bovine blood cells and peritoneal and lung macrophages were exposed in vitro to parainfluenza-3 (PI-3) virus. Residual nonadsorbed PI-3 virus (expressed in percentage of input virus) in the supernate of the various cell fractions 1 hour after incubation at 37 C was as follows: lung macrophages, 11%; peritoneal macrophages, 59%; monocytes, 26%; RBC, 14%; lymphocytes, 28%; and polymorphonuclear cells (PMN), 63%. Lung macrophages, monocytes, lymphocytes, and PMN were monitored over a 72-hour period for hemadsorption of chicken RBC. Hemadsorption increased for lung macrophages and monocytes, whereas it decreased for lymphocytes and PMN. Infective virus could not be recovered from PMN, RBC, lymphocytes, or monocytes for more than 24 hours after PI-3 infection. Recovery of infective PI-3 virus from infected peritoneal and lung macrophages extended over 4 to 8 days, respectively.     

硒化大蒜多糖对小鼠腹腔巨噬细胞功能的影响

【目的】研究硒化大蒜多糖(sGPS)对小鼠腹腔巨噬细胞功能的影响,以期为硒化大蒜多糖的作用挖掘和临床应用提供依据。【方法】依次通过分离、纯化得到大蒜多糖(GPS),并经硝酸-亚硒酸钠硒化修饰得到sGPS₃、GPS₅和sGPS₆。以小鼠腹腔巨噬细胞为研究对象,用6.25、12.5、25、50、100μg/mL sGPS₃、GPS₅、sGPS₆及10μg/mL脂多糖(LPS组)处理小鼠腹腔巨噬细胞48 h,同时设置不加药物的细胞为对照组,用中性红法测定其吞噬功能,CCK-8法测定其増殖能力,筛选出活性最好的硒化大蒜多糖;然后用ELISA法检测活性最好的硒化大蒜多糖对巨噬细胞上清液中一氧化氮(NO)、肿瘤坏死因子-α(TNF-α)、干扰素-γ(IFN-γ)、白介素-1β(IL-1β)、白介素-6(IL-6)、白介素-12(IL-12)含量的影响;再通过小鼠碳廓清实验、小鼠腹腔巨噬细胞吞噬鸡红细胞实验测定空白对照组(生理盐水)及高(2 mg/mL)、中(1 mg/...     

Monoclonal antibodies against porcine macrophages

Two mouse monoclonal antibodies (MAB) (clones 2G6 and 2B10) directed against porcine macrophages are described that are suitable for use in immunohistochemistry, FACS analysis and western blot. As immunogen, porcine cells from bronchoalveolar lavage (BAL) were used. The MABs obtained belonged to the mouse IgG1 subclass. The molecular weights of the corresponding antigens were detected by western blot under non-reducing conditions (2G6: 140-150kDa; 2B10: 140-145kDa). For specificity screening, porcine snap-frozen tissues of lung, lung lymph node, tonsil, spleen, thymus, brain, liver, gut and kidney were used. The MABs were able to identify cell populations of the mononuclear phagocytic system in these organs. While MAB 2G6 detected tissue macrophages (sinusoidal lymph node macrophages, red pulp spleen macrophages, Kupffer cells, Langerhans cells, thymus macrophages, macrophages of lung and macrophages of kidney), MAB 2B10 stained cells scattered in the lymph node (subsinusoidal, interfollicular and follicular macrophages) and in the lung interstitium. Additionally, it showed reactivity with Kupffer cells, spleen and kidney macrophages. An immunoreactivity of the MABs could be established also for human but not for bovine and avian macrophages. By flow cytometric analysis, MAB 2B10 reacted with a subpopulation of BAL and peritoneal cells. Antibody 2G6 detected macrophages of the BAL and the peritoneal fluid as well as peripheral blood monocytes.     

Comparison of in vitro replication and cytopathology caused by strains of canine distemper virus of vaccine and field origin

Three biological properties of canine distemper virus were examined to determine if any would consistently differentiate field from vaccine strains of the virus. The properties were the ability to (1) infect macrophages and epithelial cells, (2) produce distinct cytopathologic effect in alveolar and peritoneal macrophages and Vero cells, and (3) produce pocks on the chorioallantoic membrane of embryonated chicken eggs. Four vaccine strains and 5 field isolates were used in the study. The 5 field isolates were obtained directly from canine tissues. Of the 3 properties studied, only the comparison of the ability of the viruses to infect macrophages and epithelial cells was a consistent marker of virus origin. Virulent field isolates would only infect macrophage cultures, whereas the vaccine strains infected both types of cells. One avirulent field isolate from a case of old dog encephalitis reacted more like a vaccine strain by infecting both cell types.     

Infectious bursal disease virus (IBDV) induces apoptosis in chicken B cells

The ability of infectious bursal disease virus (IBDV) serotypes 1 and 2, and the role of VP4 of both serotypes as well as the capacity of three IBDV intermediate serotype 1-specific vaccine strains to induce apoptosis in a chicken B-lymphocyte cell line, DT40, were investigated using the TUNEL technique. It was observed that IBDV serotype 1 infected the DT40 cell line and directly induced apoptosis. In contrast, the non-pathogenic serotype 2 neither infected nor induced apoptosis, but was able to reduce the serotype 1-induced apoptosis when the two viruses were present in combination. VP4 of both serotypes did not induce apoptosis. IBDV VP2 of serotype 2 induced apoptosis in the same proportion and intensity as VP2 of serotype 1. IBDV intermediate vaccines varied in their ability to induce apoptosis in the DT40 cell line, which was also decreased-delayed in presence of serotype 2 IBDV. We hypothesize that both serotypes compete for the same receptor in DT-40 cells, and suggest that IBDV-induced apoptosis is a multistep process involving virus replication, protein expression, and release of virions.     

Bovine respiratory syncytial virus can induce apoptosis in MDBK cultured cells

Bovine respiratory syncytial virus (BRSV) is a major cause of respiratory disease in calves. BRSV infection is associated with epithelial cell death and inflammation. Over the past few years, a growing number of viruses have been found to induce apoptosis. In order to determine the ability of BRSV to induce apoptosis, we studied the effect of BRSV infection in cultured MDBK cells. We used ligation-mediated PCR assay to detect specific blunt-end cellular DNA fragments produced by cellular endonucleases cleaving the genomic DNA between the nucleosomes during apoptosis. We found that BRSV infection resulted in apoptosis in MDBK cells. This data demonstrates for the first time that BRSV can induce apoptosis. This data also may contribute to delineate the mechanisms that regulate tissue injury and potential lung repair following BRSV infection.     

Cell-mediated immune protection in chickens against Pasteurella multocida

Immune protection by cellular immunity in chickens against Pasteurella multocida was investigated by in vivo and in vitro experiments using spleen cells and culture supernatants of immunised chickens. Intraperitoneal or intravenous transfer of immune splenic cells into normal chickens induced transmission of an as effective protection as that exhibited in immunised chickens. Immune protection was also obtained by intravenous treatment of chickens with culture supernatant fluid from immune splenic cells of hormonally bursectomised chickens. The in vitro experiment showed that intracellular bacterial proliferation was inhibited in peritoneal macrophages from immunised chickens, or from normal chickens sensitised with culture supernatant fluid of immune splenic cells, and the macrophages were protected from disruption by infection. Peritoneal macrophages sensitised with culture supernatant fluid from unimmunised splenic cells, or peritoneal macrophages from unimmunised chickens, allowed considerable intracellular proliferation of bacteria with almost complete breakdown of the macrophages within 24 hours after bacterial challenge. These data suggest that the protective immunity of chickens against P multocida was dependent on cell-mediated immunity by mediators such as the macrophage activating factor from T lymphocytes.     

Role of macrophages in the expression of immune responses

Cells of the mononuclear phagocyte system have a crucial role as affector and effector cells in the body's defense against foreign cells and microorganisms. Macrophages function as the first line of defense via phagocytosis or opsonic phagocytosis as early as the promonocytic stage of their development. Macrophages act as affector cells via antigen presentation to lymphocytes, and they participate in the activation of T and B lymphocytes through the secretion of lymphostimulatory substances (monokines). In the cycle of reciprocal interactions, macrophages are themselves being activated via the secretory products of the lymphocytes--the lymphokines. Activated macrophages are endowed with effector functions exerted by their tumoricidal, microbicidal, and suppressor activities. Undoubtedly, additional research will enhance the importance and application of this unique cell type with multiple functions.     

B-congenic chickens differ in macrophage inflammatory responses

The influence of the chicken major histocompatibility (B) complex (MHC) on monocyte and macrophage recruitment and activation was examined using fully developed 15I5-B congenic White Leghorn lines (ten backcross generations). The phagocytic activity of Sephadex-elicited peritoneal macrophages for sheep red blood cells (SRBCs) was highest in lines 15.7-B2 and 15.P-B13 and lowest in 15.15I-B5 and 15.N-B21. The same pattern of phagocytic activity was obtained when LPS (E. coli) was used as the in vivo elicitor-activator of peritoneal macrophages. Lines with B2 and B13 haplotypes had elevated percentages of phagocytic macrophages and a higher internalization activity per cell than did B5 and B21 congenic chickens. Differential peritoneal macrophage function between congenic lines was further supported by quantitation of superoxide anion release. B2 and B13 haplotypes were associated with high activity in contrast with B5, which was low, and 15I5 (B15) and B21 which were intermediate for superoxide anion release by macrophages. In vitro activation of blood monocytes with LPS resulted in similar line differences for SRBC phagocytic activity as were observed with in vivo Sephadex and LPS activation. In contrast, chemotaxis of blood mononuclear leukocytes to f-met-leu-phe produced a reciprocal response pattern among the haplotypes. Cells from lines with haplotypes B5 and B21 were superior to those of B2, B13, and B15 congenic lines in their directed migration towards this chemoattractant. All functional differences occurred despite similarities among lines in the cellular profiles of both elicited peritoneal exudate cells and isolated blood mononuclear cells.     

猪繁殖与呼吸综合征病毒实验感染SPF猪外周血单核细胞和肺泡巨噬细胞早期凋亡细胞的观察

采用Ａｎｎｅｘｉｎ Ｖ－ＦＩＴＣ／ＰＩ双染色法,用流式细胞仪检测了猪繁殖与呼吸综合征病毒（ＰＲＲＳＶ）实验感染ＳＰＦ猪不同时期外周血单核细胞和肺泡巨噬细胞感染Ａｎｎｅｘｉｎ Ｖ－ＦＩＴＣ＾＋／ＰＩ＾－细胞群（早期凋亡细胞群）。结果显示,ＰＲＲＳＶ感染猪外周血单核细胞和肺泡巨噬细胞Ａｎｎｅｘｉｎ Ｖ－ＦＩＴＣ＾＋／ＰＩ＾－细胞群的表达率均明显高于正常对照猪,感染后２４ｈ表达率达最高值。     

Tilmicosin-induced bovine neutrophil apoptosis is cell-specific and downregulates spontaneous LTB4 synthesis without increasing Fas expression

The pathology of bacterial pneumonia, such as seen in the bovine lung infected with Mannheimia haemolytica, is due to pathogen virulence factors and to inflammation initiated by the host. Tilmicosin is a macrolide effective in treating bacterial pneumonia and recent findings suggest that this antibiotic may provide anti-inflammatory benefits by inducing polymorphonuclear neutrophilic leukocyte (PMN) apoptosis. Using an in vitro bovine system, we examined the cell-specificity of tilmicosin, characterized the changes in spontaneous leukotriene B4 (LTB4) synthesis by PMN exposed to the macrolide, and assessed its effects on PMN Fas expression. Previous findings demonstrated that tilmicosin is able to induce PMN apoptosis. These results were confirmed in this study by the Annexin-V staining of externalized phosphatidylserine and the analysis with flow cytometry. The cell-specificity of tilmicosin was assessed by quantification of apoptosis in bovine PMN, mononuclear leukocytes, monocyte-derived macrophages, endothelial cells, epithelial cells, and fibroblasts cultured with the macrolide. The effect of tilmicosin on spontaneous LTB4 production by PMN was evaluated via an enzyme-linked immunosorbent assay. Finally, the mechanisms of tilmicosin-induced PMN apoptosis were examined by assessing the effects of tilmicosin on surface Fas expression on PMN. Tilmicosin-induced apoptosis was found to be at least partially cell-specific, as PMN were the only cell type tested to die via apoptosis in response to incubation with tilmicosin. PMN incubated with tilmicosin under conditions that induce apoptosis spontaneously produced less LTB4, but did not exhibit altered Fas expression. In conclusion, tilmicosin-induced apoptosis is specific to PMN, inhibits spontaneous LTB4 production, and occurs through a pathway independent of Fas upregulation.     

Discovery of natural products capable of inducing porcine host defense peptide gene expression using cell-based high throughput screening

Background: In-feed antibiotics are being phased out in livestock production worldwide. Alternatives to antibiotics are urgently needed to maintain animal health and production performance. Host defense peptides(HDPs) are known for their broad-spectrum antimicrobial and immunomodulatory capabilities. Enhancing the synthesis of endogenous HDPs represents a promising antibiotic alternative strategy to disease control and prevention.Methods: To identify natural products with an ability to stimulate the synthesis of endogenous HDPs, we performed a high-throughput screening of 1261 natural products using a newly-established stable luciferase reporter cell line known as IPEC-J2/pBD3-luc. The ability of the hit compounds to induce HDP genes in porcine IPEC-J2 intestinal epithelial cells, 3 D4/31 macrophages, and jejunal explants were verified using RT-qPCR. Augmentation of the antibacterial activity of porcine 3 D4/31 macrophages against a Gram-negative bacterium(enterotoxigenic E. coli) and a Gram-positive bacterium(Staphylococcus aureus) were further confirmed with four selected HDP-inducing compounds.Results: A total of 48 natural products with a minimum Z-score of 2.0 were identified after high-throughput screening,with 21 compounds giving at least 2-fold increase in luciferase activity in a follow-up dose-response experiment.Xanthohumol and deoxyshikonin were further found to be the most potent in inducing p BD3 m RNA expression,showing a minimum 10-fold increase in IPEC-J2, 3 D4/31 cells, and jejunal explants. Other compounds such as isorhapontigenin and calycosin also enhanced p BD3 m RNA expression by at least 10-fold in both IPEC-J2 cel s and jejunal explants, but not 3 D4/31 cells. In addition to p BD3, other porcine HDP genes such as p BD2, PG1-5, and p EP2 C were induced to different magnitudes by xanthohumol, deoxyshikonin, isorhapontigenin, and calycosin, although clear gene-and cel type-specific patterns of regulation were observed. Desirably, these four compounds had a minimum effect on the expression of several representative inflammatory cytokine genes. Furthermore, when used at HDP-inducing concentrations, these compounds showed no obvious direct antibacterial activity, but significantly augmented the antibacterial activity of 3 D4/31 macrophages(P 0.05) against both Gram-negative and Gram-positive bacteria.Conclusions: Our results indicate that these newly-identified natural HDP-inducing compounds have the potential to be developed as novel alternatives to antibiotics for prophylactic and therapeutic treatment of infectious diseases in livestock production.