Cytokines and Ly-1 (B1) B Cells

In an attempt to elucidate the possible role of cytokines in autocrine growth of Ly-1 + B cells, and the role of this subset of B cells in immune regulation, both in normal and diseased hosts, we have performed a systematic analysis of cytokine production by a series of mouse Ly-1⁺ B lymphomas, as well as normal peritoneal Ly-1⁺ and conventional B cells. The lymphomas all express TGF-β, and some express IL-3 and IL-4. We observed that both the lymphomas and the peritoneal cells produce TNF-α, TNF-β and IL-6. Another cytokine, IL-10, is produced predominantly by peritoneal Ly-1⁺ B cells from healthy mice and by Ly-1⁺ B lymphomas, but not by conventional B cells. As IL-10 regulates the production of monokines and a subset of T-cell derived cytokines, our results suggest a broad immunoregulatory role for Ly-1 B cells. To complement these studies we have also examined the responses of Ly-1 B cells to mitogens and cytokines previously shown to stimulate conventional B cells. In summary, Ly-1 B cells, in contrast to conventional B cells do not respond to anti-Ig antibodies, even in the presence of IL-4. They do respond to LPS, and this response is preferentially enhanced by IL-5, and marginally enhanced by IL-3. Surprisingly LPS-induced proliferation of peritoneal B cells is inhibited by IL-6 and to a greater extent by IL-10. Whether this inhibition is a result of differentiation into Ig secreting cells is currently being evaluated. We discuss our findings in terms of the potential of Ly-1 B cells to regulate their own development and the immunocompetence of other cells.     

Developmental Origins,Specificities and Immunoglobulin Gene Biases of Murine Ly-1 B Cells

Ly-1 B cells in mouse show numerous phenotypic and functional features that distinguish them from the bulk of IgD^high/Ly-1^? B cells. Their association with autoantibody production and the presence of Ly-1 on a group of murine B lymphomas that also exhibit certain specificities enriched in the normal population has stimulated continuing interest in this population. We have taken two approaches in our investigations of these cells: 1) defining the origins of Ly-1 B cells (the “lineage question”); and 2) studying the expression of particular specificities and associated immunoglobulin V genes enriched in this population. In this review we present the experimental background that supports our current understanding of Ly-1 B cells as the remnant of a fetal B cell differentiation pathway and suggest that the selection of cells from this fetal/neonatal population into the adult long-lived pool results in the over-expression of certain germline-encoded autoreactivities, such as antibody to bromelain-treated mouse red blood cells and intact thymocytes.     

In vivo neutralization of hepatitis B virus infection by an anti-preS1 humanized antibody in chimpanzees

Previously, we generated a murine monoclonal antibody (mAb), KR127, that recognizes amino acids (aa) 37-45 of the preS1 of hepatitis B virus (HBV). In this study, we have constructed a humanized version of KR127 and evaluated its HBV-neutralizing activity in chimpanzees. A study chimpanzee was given a single intravenous dose of the humanized antibody, followed by intravenous challenge with adr subtype of wild type HBV, while a control chimpanzee was only challenged with the virus. The result showed that the study chimpanzee did not develop HBV infection during 1 year, while the control chimpanzee was infected, indicating that the humanized antibody exhibited in vivo virus-neutralizing activity and thus protected the chimpanzee from HBV infection. In addition, the humanized antibody bound to the preS1 of all subtypes of HBV. We first demonstrate that an anti-preS1 mAb can neutralize HBV infection in vivo. This humanized antibody will be useful for the immunoprophylaxis of HBV infection.     

In vivo activation of murine natural killer cell functions by human recombinant DNA interleukin 2

Intraperitoneal (i.p.) injections of purified human recombinant DNA (rDNA)-interleukin 2 (IL 2) resulted in in vivo activation of local natural killer (NK) cell activities in wild-type and congenitally athymic mice. NK cells were identified by short-term cytotoxicity assays against YAC tumor targets and by cell-surface phenotyping. The magnitude of the cytolytic responses was dependent on the IL 2 dose (greater than or equal to 0.1 microgram per injection) and the time period of treatment (the maximum response was on days 3 to 4 after daily treatment). In vivo application of antisera against the murine NK marker asialo GM1 (asGM1) and against interferon-alpha/beta and -gamma (IFN) significantly inhibited NK cell activation. Limiting dilution analysis revealed high frequencies (up to 1 in 1.8 X 10(3)) of in vitro IL 2 reactive mononuclear cells among the peritoneal exudate cells (PEC) of normal mice. rDNA-IL 2 activated non-adherent PEC to proliferate. The majority of these cultures also displayed cytotoxicity against YAC targets. No exogenous IFN was required for either response. Endogenous IFN production did not appear to play an important role for induction of cytotoxicity in this system either. Only a minority of cultures produced measurable levels of IFN without showing excessive cytotoxic activity. In vivo IL 2 treatment resulted in a rapid increase of the total numbers and frequencies of the IL 2 reactive PEC. Hence, IL 2 alone was apparently sufficient for in vitro activation of NK-like activities, whereas IFN-induction by IL 2 was required for in vivo elicitation of similar responses in perhaps the same cell populations.     

Expression of the bovine papillomavirus type 1 E5B gene reveals a protein-protein interaction of the E5A and E5B gene products

The bovine papillomavirus type 1 (BPV-1) genome has been shown to contain a small open-reading frame designated E5B (nucleotides 4013-4167) which is predicted to encode a hydrophobic, 52 amino acid protein. In order to detect and characterize the E5B protein, an 18 nucleotide sequence encoding a 6 amino acid epitope was added to the 3' end of the E5B open-reading frame which was then expressed in COS-1 cells using a SV40 vector. Immunoprecipitation, immunofluorescence, and cell fractionation studies identified the E5B protein as a 4-kDa protein and localized it primarily to membranes of the endoplasmic reticulum and nucleus. Unlike the E5A protein of BPV-1, E5B did not form dimers (despite containing a cysteine residue) or form complexes with growth factor receptors such as the PDGF receptor or erb B-2 receptor. Interestingly, the E5B protein formed physical complexes with the hydrophobic E5A oncoprotein, apparently via transmembrane interactions. Additionally, expression of E5B inhibited the transforming capability of BPV-1 E5A. These observations suggest that the expression of this viral protein may play a significant role in BPV/host cell interactions.     

In situ expression of B7/BB1 on antigenpresenting cells and activated B cells: an immunohistochemical study

B7/BB1 is a physiological ligand for CD28, a receptor expressedon a major subset of T lymphocytes. B7/BB1 has been shown tobe expressed on human blood dendritic cells and on in vitroactivated (but not resting) B cells and monocytes. Llgatlonof CD28 with B7/BB1 upregulates cytoklne production and preventsthe Induction of anergy in T cells activated through TCR/CD3.We examined the in situ expression of B7/BB1 by immunohlstochemlstrywith a novel mAb B7-24. Dendritic cells in skin (Langerhanscells), lymph node sinuses (veiled cells), and T cell zonesof spleen and lymph nodes (Interdlgltatlng dendritic cells)were strongly positive for B7/BB1. B7/BB1 was also present onfetal thymus dendritic cells located at the cortlco-medullarJunction and the medulla, but absent in normal adult thymuses.Resident macrophages and endothellal cells did not stain, butin granulomatous inflammations B7/BB1 was found on macrophagesand epltheloid cells. A subset of B immunoblasts and of germinalcenter B cells In lymph node and spleen was also found to expressB7/BB1. Our findings on the distribution of B7/BB1 expressionIn tissues, in particular its expression on professional antigen-presentingcells, further substantiate the putative co-stlmulatory roleof B7/BB1 in T cell activation in vivo. The presence of B7/BB1in fetal but not adult thymlc medulla suggests a role for B7/BB1in thymocyte maturation.     

IL-17RA-signaling in Lgr5+ intestinal stem cells induces expression of transcription factor ATOH1 to promote secretory cell lineage commitment

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Administration of interleukin -5 or -10 activates peritoneal B-1 cells and induces autoimmune hemolytic anemia in anti-erythrocyte autoantibody-transgenic mice

Activation mechanisms of B-1 (Ly-1 B) cells have been suggested to be different from those of conventional B cells. To assess the role of various interleukins (IL) in the activation of B-1 cells, we injected IL-4, IL-5 or IL-10 into nonanemic anti-red blood cells (RBC) autoantibody-transgenic mice, in which conventional B cells are clonally deleted but peritoneal B-1 cells persist without secreting Ig. Intraperitoneal or intramuscular injection of IL-5 or IL-10, but not IL-4, increased the number of antibody-producing peritoneal B-1 cells by four- to five-fold, resulting in increased anti-RBC serum autoantibody and induction of hemolytic anemia. These results suggest that IL-5 or IL-10 may play an important role in the terminal differentiation of B-1 cells into antibody-producing cells in vivo.     

Maternal effects in infant and adult phenotypes of 5HT1A and 5HT1B receptor knockout mice

The influence of the pre- and postweaning maternal environment on the offspring's phenotype was examined in 5-HT1A and 5-HT1B receptor knockout mice (KO1A and KO1B, respectively). We have previously shown that, when born to and raised by homozygous dams of the same genotype, adult KO1A are more anxious than wild-type (WT) mice, and adult KO1B are hyperactive and slightly less anxious than WT mice. We extend our studies here to the behavioral results of the offspring's own genotype, when the dam's genotype is constant, and the effects of the dam's genotype when the offspring's genotype is constant. In Experiments 1 and 2, KO1A-/- pups produced less ultrasonic vocalizations (USV) than controls in an isolation test on postnatal Day 7 when born to and reared by KO1A dams, either -/- or +/-. Heterozygous F1 pups reared by KO1A-/- dams produced more USV and were less anxious in the plus-maze at 2 to 3 months of age than F1 pups born to and reared by WT dams (Experiment 3). F1 pups reared by KO1B-/- dams produced less USV and were more anxious in the plus-maze than F1 pups reared by WT dams (Experiment 4). The results support a role for maternal effects that may comprise direct effects such as the dam's behavior and nutritional care of the pup, and possibly more complex indirect effects through the establishment of idiosyncratic dam-pup dyadic interactions. We recommend that breeding techniques that rely on same genotype (mutant-mutant or WT-WT) breeding pairs not be used to generate offspring when the focus of research is the study of gene function, but rather when familial effects need to be studied.     

Association between ICAM-1 expression and metastatic capacity of murine B-cell hybridomas

We previously reported that a derivative of the interleukin-6 (IL-6)-dependent B9 B-cell hybridoma (B9/LPNUIL) constitutively expressing an interleukin-1a (IL-1a) gene introduced by retrovirus-mediated gene transfer preferentially metastasized to bone marrow following intravenous injection into unirradiated syngeneic BALB/c mice. B9/LPNUIL cells recovered from the femoral marrow of a recipient with hind limb paralysis (denoted B9/BMl) retained their IL-6-dependency yet displayed enhanced metastatic capacity during serial transplantation in vivo. In contrast, autonomously-growing B9 variants spontaneously arising in vitro or IL-6-independent B9 derivatives created by infection with recombinant IL-6 retroviruses rarely gave rise to experimental metastases in syngeneic BALB/c or nude mice. Examination of cell adhesion molecule profiles by immunofluorescence flow cytometry has revealed high levels of CD44, moderate levels of VLA-4 and low levels of LFA-1 on all B9-series cells. By comparison, ICAM-1 expression was significantly elevated on B9/BMl cells, with independent isolates stably expressing about 4-fold higher levels which were paralleled by corresponding increases in the steady-state levels of ICAM-1 mRNA. l-Selectin was not expressed by any of the cell lines. Despite higher ICAM-1 levels, cell aggregation assays revealed that LFA-1-ICAM-1 adhesive interactions were not involved in the homotypic adhesion of B9/BM1 cells but rather that binding of CD44 to endogenously-synthesized hyaluronan was responsible. Furthermore, B9/BM1 cells expressing high levels of ICAM-1 were found to be less susceptible to cytolysis by natural killer (NK) cells than their weakly metastatic or nonmetastatic counterparts.     

Purification and characterization of mouse mast cell proteinase-2 and the differential expression and release of mouse mast cell proteinase-1 and -2 in vivo

BACKGROUND: Gastrointestinal nematode infection is associated with mucosal mast cell (MMC) hyperplasia. In the mouse, this is accompanied by the release of substantial quantities of the chymase mouse mast cell proteinase-1 (mMCP-1) into the gut lumen and peripheral bloodstream. Expression of mMCP-1 is largely restricted to intraepithelial MMC and is thought to play a role in the regulation of epithelial permeability. MMCs also express mouse mast cell proteinase-2 (mMCP-2), but less is known about the expression or biological function of this proteinase. OBJECTIVES: (1) To purify and characterize mMCP-2. (2) To compare the expression and release of mMCP-2 and mMCP-1 in vivo using specific antibodies. METHODS: Bone marrow-derived mast cells (mBMMCs) were generated from mMCP-1(-/-) BALB/c mice. mMCP-2 was purified, characterized and used to generate rat and sheep polyclonal antibodies. The expression and systemic release of mMCP-1 and -2 were compared in vivo by immunohistochemistry and ELISA. RESULTS: mMCP-2 was successfully purified from mMCP-1(-/-) mBMMC and its identity confirmed by N-terminal amino acid sequencing. mMCP-2 bound [3H]-labelled DFP, indicating the presence of an active serine proteinase catalytic site, but showed little evidence of chymotryptic activity. MMC expressed comparable levels of mMCP-1 and -2 in the jejunum but not in the gastric mucosa, where mMCP-2 was more abundant. Expression of both proteinases increased substantially during primary Nippostrongylus brasiliensis infection and this was accompanied by a substantial increase in peripheral blood levels of mMCP-1 (70 microg/mL on day 12). By contrast, mMCP-2 was not detected in the serum of uninfected mice and only increased to approximately 25 ng/mL on day 12. CONCLUSION: As in the case of mMCP-1, mMCP-2 expression is restricted to MMC. However, mMCP-2 lacks chymase activity, is expressed at higher levels in gastric MMC and appears to be differentially released into the peripheral bloodstream.     

In vivo neutralization of α4 and β7 integrins inhibits eosinophil trafficking and prevents lung injury during tropical pulmonary eosinophilia in mice

Integrins regulate leukocyte trafficking during homeostasis and inflammatory conditions. However, the role of α4 and β7 integrins in guiding eosinophil transmigration into the lungs during filarial manifestation of Tropical Pulmonary Eosinophilia (TPE) has not been explored. In this study, mice exhibiting TPE manifestations were administered with in vivo neutralizing antibodies against integrins α4 and β7 or their combination and immuno‐pathological parameters were evaluated. Results show an intact lung barrier, significantly lower lung inflammation and reduced eosinophil counts in the Bronchoalveolar lavage fluid and lungs of mice receiving anti‐α4⁺β7 treatment. Reduced eosinophil peroxidase and β‐hexosaminidase activity, downregulation of inflammatory genes, lower production of inflammatory lipid intermediates like prostaglandins E2 and D2, leukotriene B4 and cysteinyl leukotrienes were also noted in anti‐α4⁺β7 treated mice. Reduced accumulation of central memory, effector memory, regulatory T cells and lower production of IL‐4, IL‐5, and TGF‐β were other cardinal features of anti‐α4⁺β7 treated mice lungs. Flow cytometry‐sorted lung eosinophils from anti‐α4⁺β7 treated mice showed higher apoptotic potential, downregulated anti‐apoptotic gene Bcl‐2, and exhibited reduced F‐actin polymerization and calcium influx as compared to IgG controls. In summary, neutralization of α4⁺β7 integrins impairs the transmigration, activation and survival of eosinophils and reduces TPE induced pathology in mice lungs.     

Effects of the selective 5-HT1B agonist, CGS 12066B, on sleep/waking stages and EEG power spectrum in rats

SUMMARY  Sleep/waking stages and EEG power spectra were studied in rats for 8 h following intraperitoneal administration of CGS 12066B, a selective 5-HT_1B agonist. Waking was increased and rapid eye movement (REM) sleep decreased in a dose-dependent manner. Total slow-wave sleep (TSWS) was reduced, but only in the first 2 h period. The latencies to REM sleep and stable sleep were increased dose-dependently. The drug also induced profound behavioural changes that may account for some of the sleep/waking changes. EEG power densities in waking and TSWS were reduced dose-dependently from 7 to 20 Hz after CGS 12066B, suggesting a tendency towards general deactivation. The increase in waking together with a general deactivation suggest complex effects of CGS 12066B on the sleep/waking axis.     

In vivo expression of recombinant pregnancy-specific glycoprotein 1a induces alternative activation of monocytes and enhances Th2-type immune response

It has been proposed that pregnancy-specific factors could be responsible for shift the balance of cytokine profiles during maternal immune response from Th1-type reactivity into a "less-damaging" Th2-type reactivity. In the present work, we investigated the in vivo function of human pregnancy-specific glycoprotein (PSG)1a, the major variant of PSG polypeptides released into the circulation during pregnancy, on the modulation of the innate and adaptive immune response. For this, BALB/c mice were injected with a vaccinia virus-based vector harboring the human PSG1a cDNA (Vac-PSG1a) 4 days before immunization with ovalbumin (OVA) in complete Freund's adjuvant, and the early specific T cell response against OVA was evaluated 8 days post-immunization. We also studied the activation status of spleen and peritoneal monocytes/macrophages (Mo) populations from Vac-PSG1a-treated mice, and explored whether PSG1a-targeted Mo could affect the Th-type commitment by investigating their impact on the differentiation of naive T cells. Our data show that the treatment with Vac-PSG1a is able to induce a state of alternative activation on Mo. Furthermore, the generation of the immune response in the context of these alternatively activated antigen-presenting cells may shift T cell differentiation to Th2-type immunity which is more compatible with a successful pregnancy.     

Genetic association of polymorphisms at the intergenic region between PRDM1 and ATG5 with hepatitis B virus infection in Han Chinese patients

Chronic hepatitis B virus (HBV) infection is related to chronic hepatitis, cirrhosis, and hepatocellular carcinoma (HCC), and the interplay between the virus and host immune response leads to different outcomes of the infection. PR domain zinc finger protein 1 (PRDM1) and autophagy-related protein 5 (ATG5) are involved in immune response and HBV infection. An intergenic region between PRDM1 and ATG5 (PRDM1-ATG5 region) has been identified, and single-nucleotide polymorphisms (SNPs) in this region were shown to be involved in immune regulation. This study investigated the functionally relevant rs548234, rs6937876, and rs6568431 polymorphisms at the PRDM1-ATG5 region in a Han Chinese population (403 patients with chronic HBV infection [171 chronic hepatitis, 119 cirrhosis, and 113 HCC], 70 infection resolvers, and 196 healthy controls). The frequencies of the rs6568431 allele A in HBV patients (P = .005) and genotype CA in infection resolvers (P = .005) were significantly higher than in healthy controls. In the dominant model, HCC patients had significantly higher frequencies of rs548234 genotypes CC + TC than cirrhosis patients (P = .009). Rs548234 was an independent factor for HCC in comparison with either cirrhosis (P = .005) or all chronic HBV infection without HCC (P = .018). Functional annotation showed evidence of the role of the SNPs in gene regulation. In conclusion, through this study it is revealed for the first time that rs6568431 may be associated with susceptibility to HBV infection and that rs548234 may be associated with HCC risk in chronic HBV infection, supporting the presence of HBV-related disease-causing regulatory polymorphisms in the PRDM1-ATG5 intergenic region.     

Knockdown of KDM1B inhibits cell proliferation and induces apoptosis of pancreatic cancer cells

Pancreatic cancer (PC) is one of the common malignant tumors in digestive tract with a high fatality rate. The oncogenic role of lysine-specific demethylase1 (LSD1/KDM1?A) has been well recognized in PC. While, the role of its homolog LSD2 (KDM1B) in regulating PC progression is poorly understood. In this study, we attempted to evaluate the functional role of KDM1B in PC cells. The expression of KDM1B was detected by immunohistochemistry and immunoblotting in PC tissues and cells. Lentivirus-mediated shRNA was applied to silence KDM1B in PANC-1 and SW1990 cells. Cell proliferation was measured by MTT and Celigo assay. Cell apoptosis was determined by both Caspase-Glo^®3/7 assay and Flow cytometry. Intracellular signaling molecules were detected using a PathScan intracellular signaling array kit. In this study, we found KDM1B was highly expressed in PC tissues compared to paracancerous tissues. Moreover, elevated expression of KDM1B was detected in PC cell lines (BxPC-3, CFPAC-1, PANC-1 and SW1990) as compared with a normal human pancreatic duct epithelial cell line (HPDE6-C7). Further investigations revealed that KDM1B knockdown significantly inhibited PC cell proliferation. Furthermore, the apoptosis of PANC-1 and SW1990 cells was significantly increased after KDM1B knockdown. Notably, the activations of p-ERK1/2, p-Smad2, p-p53, cleaved PARP, cleaved Caspase-3, cleaved Caspase-7, p-eIF2a and Survivin were promoted by KDM1B knockdown, while IkBa was suppressed. Taken together, our findings provided new insights into the critical and multifaceted roles of KDM1B in the regulation of cell proliferation and apoptosis, and offered a potentially novel target in preventing the progression of PC.     

人参皂甙Rb1通过Rho/Rho激酶通路影响低氧诱导大鼠肺动脉平滑肌细胞增殖及SERT和5-HT_(1B)R表达

目的:观察人参皂甙Rb1对低氧性大鼠肺动脉平滑肌细胞(PASMCs)5-羟色胺转运体(SERT)和5-羟色胺1B受体(5-HT_(1B)R)表达及细胞增殖的影响,并探讨Rho/Rho激酶通路在其中的作用。方法:分离并培养健康雄性SD大鼠PASMCs,随机分为常氧组(normal组)、低氧组(hypoxia组)以及低氧加50、100和200 mg/L人参皂甙Rb1组(HR50、HR100和HR200组),采用CCK-8、Brd U结合流式细胞术、Western blot及RT-PCR等方法,观察大鼠PASMCs的增殖程度以及SERT和5-HT_(1B)R的mRNA和蛋白表达变化;另取PASMCs分为normal组、hypoxia组、HR200组和低氧加Y-27632组(HY组),检测Rho激酶(ROCK1)mRNA表达和肌球蛋白磷酸酶目标亚单位1(MYPT1)磷酸化水平。结果:与normal组比较,hypoxia组PASMCs增殖明显(P0.01);与hypoxia组比较,人参皂甙Rb1可明显抑制PASMCs的增殖(P0.01),并浓度依赖性抑制SERT和5-HT_(1B)R的mRNA与蛋白表达(P0.05);HR200组可明显抑制ROCK1的mRNA表达和MYPT1磷酸化水平(P0.01),与HY组相比较,差异无统计学显著性。结论:低氧能诱导大鼠PASMCs增殖,上调SERT和5-HT_(1B)R表达;人参皂甙Rb1能浓度依赖性抑制这种作用,其机制可能与抑制Rho/Rho激酶通路表达有关。