淋病奈瑟菌对hCD46基因转染COS-1细胞的黏附

为研究hCD46在菌毛介导的淋病奈瑟菌宿主特异性黏附过程中的作用。用连接PCR技术扩增出启动子与cDNA相连的hCD46小基因,并将其置换出pcDNA3.1载体中的CMV启动子,构建成重组真核表达载体pCD46。转染COS-1细胞后用间接免疫荧光法检测到hCD46 cDNA在其自身启动子的指导下可在COS-1细胞膜表面有效表达,Western blotting检测表明表达产物的分子正确,用流式细胞术分选表达hCD46的基因转染细胞COS-1-46。细菌黏附实验显示菌毛阳性淋病奈瑟菌临床分离株对COS-1-46细胞有较强的黏附性。提示hCD46是菌毛介导的淋病奈瑟菌特异性黏附于人黏膜细胞的一种重要受体,hCD46小基因可用于淋病奈瑟菌感染转基因小鼠模型的制备。     

Tyrosine phosphorylation of a novel 100-kDa protein coupled to CD28 in resting human T cells is enhanced by a signal through TCR/CD3 complex

For T cell activation, two signals are required, i.e., a T cell receptor (TCR)/CD3-mediated main signal and a CD28-mediated costimulatory signal. CD28 binds to its ligand (CD80 or CD86) and transduces the most important costimulatory signal. The cytoplasmic domain of the CD28 molecule, composed of 41 amino acids, does not contain any intrinsic enzyme activity. The cytoplasmic domain of CD28 is remarkably conserved among species and is associated with a number of signaling molecules that affect the main signal. We report here that a tyrosine phosphorylated 100-kDa protein (ppl00) was coupled to the CD28 cytoplasmic domain in Jurkat and human peripheral T cells. The pp100 was distinguished from other CD28 associated molecules such as Vav, STAT5, PI 3-kinase, Valosin-containing protein (VCP), Nucleolin, Gab2 (Grb2-associated binding protein 2), and STAT6. The tyrosine phosphorylation of pp100 coprecipitated with CD28 was enhanced by CD3 stimulation by the specific antibody, tyrosine phosphatase inhibitor and PKC activator. Tyrosine phosphorylation of pp100 was attenuated by the prior addition of PKC inhibitor. These findings indicate that pp100 is a novel tyrosine phosphorylated protein coupled to CD28 under continuous control of tyrosine phosphatases and might play a role in T cell activation augmented by a TCR/CD3-mediated main signal.     

Retinoic acid treated HL60 cells express CEACAM1 (CD66a) and phagocytose Neisseria gonorrhoeae

Neisseria gonorrhoeae (gonococci, GC) are phagocytosed by neutrophils through the interaction between opacity proteins (Opa) and the CEA (CD66) family of antigens. In order to study this interaction, we used the human myeloid leukemia HL60 cell line, which differentiates into granulocyte-like cells upon treatment with dimethylsulfoxide (DMSO) or retinoic acid (RA). We found that RA-, but not DMSO- or untreated-HL60 cells, can phagocytose OpaI-expressing gonococci as well as Escherichia coli. The interaction of OpaI E. coli with RA-treated HL60 cells was inhibited by antibodies against CEACAM1. Phagocytosis of OpaI E. coli was found to be a result of the expression of CEACAM1 in RA-treated HL60 cells. Our results indicate that the level of expression of CEACAM1 in HL60 cells can be regulated by treatment with RA in a differentiation-dependent manner, and that this is important for phagocytosis of OpaI-expressing gonococci or E. coli.     

Identification of mouse NMDA receptor subunit NR2A C-terminal tyrosine sites phosphorylated by coexpression with v-Src

The protein tyrosine kinase Src is known to regulate NMDA receptors in native neurons. While NR2A, NR2B and NR2D are known to be phosphorylated on tyrosine residues, the exact sites have remained unidentified. Immunoprecipitation of NMDA receptor subunits followed by western blotting was used to analyze the state of tyrosine phosphorylation of recombinant NMDA receptor subunits expressed in HEK293 cells. Using antiphosphotyrosine antibody PY20, we find that on expression in HEK cells, v-Src and Fyn cause detectable tyrosine phosphorylation only of NR2A. Because a stronger signal was produced by the constitutively active v-Src, the general region of v-Src phosphorylation was delimited by expression of a series of truncation mutants of NR2A. Site-directed mutagenesis on candidate sites within the likely region allowed identification of three sites, Y1292, Y1325, and Y1387 that account for a significant fraction of the total PY20 signal. Two of these sites, Y1292 and Y1387, were suggested to control current modulation by Src in previous studies of HEK cells expressing NR1/NR2A. One of these sites, Y1325, has not yet been evaluated for effects on receptor current. A unique tyrosine site, Y1267, was shown not to be a site of detectable phosphorylation, in accordance with its Src-independent regulation of receptor currents.     

Manganese induces sustained Ser40 phosphorylation and activation of tyrosine hydroxylase in PC12 cells

Manganese (Mn²⁺) is an essential metal involved in normal functioning of a range of physiological processes. However, occupational overexposure to Mn²⁺ causes neurotoxicity. The dopaminergic system is a particular target for Mn²⁺ neurotoxicity. Tyrosine hydroxylase (TH) is the rate limiting enzyme for dopamine synthesis and is regulated acutely by phosphorylation at Ser40 and chronically by protein synthesis. In this study we used pheochromocytoma 12 cells to investigate the effects of Mn²⁺ exposure on the phosphorylation and activity of TH. Mn²⁺ treatment for 24 h caused a sustained increase in Ser40 phosphorylation and TH activity at a concentration of 100 μM, without altering the level of TH protein or PC12 cell viability. Inhibition of protein kinase A and protein kinase C and protein kinases known to be involved in sustained phosphorylation of TH in response to other stimuli did not block the effects of Mn²⁺ on Ser40 phosphorylation. A substantial increase in H₂O₂ production occurred in response to 100 μM Mn²⁺. The antioxidant Trolox^TM completely inhibited H₂O₂ production but did not block TH phosphorylation at Ser40, indicating that oxidative stress was not involved. Sustained TH phosphorylation at Ser40 and the consequent activation of TH both occurred at low concentrations of Mn²⁺ and this provides a potential new mechanism for Mn²⁺-induced neuronal action that does not involve H₂O₂-mediated cell death.     

Retinol activates tyrosine hydroxylase acutely by increasing the phosphorylation of serine40 and then serine31 in bovine adrenal chromaffin cells

Tyrosine hydroxylase is the rate-limiting enzyme in the biosynthesis of the catecholamines. It has been reported that retinol (vitamin A) modulates tyrosine hydroxylase activity by increasing its expression through the activation of the nuclear retinoid receptors. In this study, we observed that retinol also leads to an acute activation of tyrosine hydroxylase in bovine adrenal chromaffin cells and this was shown to occur via two distinct non-genomic mechanisms. In the first mechanism, retinol induced an influx in extracellular calcium, activation of protein kinase C and serine40 phosphorylation, leading to tyrosine hydroxylase activation within 15 min. This effect then declined over time. The retinol-induced rise in intracellular calcium then led to a second slower mechanism; this involved an increase in reactive oxygen species, activation of extracellular signal-regulated kinase 1/2 and serine31 phosphorylation and the maintenance of tyrosine hydroxylase activation for up to 2 h. No effects were observed with retinoic acid. These results show that retinol activates tyrosine hydroxylase via two sequential non-genomic mechanisms, which have not previously been characterized. These mechanisms are likely to operate in vivo to facilitate the stress response, especially when vitamin supplements are taken or when retinol is used as a therapeutic agent.     

Regulation of cell shape and adhesion by CD34

We previously reported that expression of CD43/leukosialin induces cell rounding and microvillus formation via inhibition of cell adhesion. Here, we found that CD34, a cell surface sialomucin and marker for hematopoietic progenitor cells, also inhibited cell adhesion and induced cell rounding and microvillus formation. Forced expression of CD34-induced cell rounding, microvillus formation, and phosphorylation of ezrin/radixin/moesin (ERM) proteins in HEK293T cells, while inhibiting integrin-mediated cell re-attachment. Furthermore, CD34+ blood cells and KG-1 cells, which express endogenous CD34 on their surface, were spherical in shape, surrounded by microvilli, and non-adherent to substrata. In addition, cleavage of O-sialomucin augmented integrin-mediated cell adhesion of KG-1 cells. These results suggest the involvement of CD34 in the inhibition of integrin-mediated cell adhesion and formation of the cell surface structure. The inhibitory function of CD34 in cell adhesion may affect cell shape organization via phosphorylation of ERM proteins. Cellular structures such as the spherical shape and microvilli of CD34+ cells may also contribute to regulation of cell adhesion.     

Regulation of hyaluronan binding by F-actin and colocalization of CD44 and phosphorylated ezrin/radixin/moesin (ERM) proteins in myeloid cells

Proinflammatory cytokines such as TNF-alpha up-regulate the expression of the cell adhesion molecule, CD44, and induce hyaluronan (HA) binding in peripheral blood monocytes (PBM). Here we show that in PBM, TNF-alpha induced cytoskeletal rearrangement, increased threonine phosphorylation of ERM proteins, and induced the redistribution and colocalization of phospho-ERM proteins (P-ERM) with CD44. In the myeloid progenitor cell line, KG1a, hyaluronan binding occurred in the pseudopod where CD44, P-ERM, and F-actin were highly localized. Hyaluronan binding correlated with high expression of both CD44 and P-ERM clustered in a single pseudopod. Disruption of polymerized actin reduced hyaluronan binding in both PBM and KG1a cells and abolished CD44 clustering and the pseudopod in KG1a cells. The pseudopod was not required for the clustering of CD44, the colocalization with P-ERM, or hyaluronan binding. However, treatment with a kinase inhibitor abolished ERM phosphorylation and reduced hyaluronan binding. Furthermore, expression of CD44 lacking the putative ERM binding site resulted in reduced hyaluronan binding. Taken together, these data suggest that CD44-mediated hyaluronan binding in human myeloid cells is regulated by P-ERM and the actin cytoskeleton.     

Heat stress induces tyrosine phosphorylation/activation of kinase Fa/GSK-3α (a human carcinoma dedifferentiation modulator) in A431 cells

Exposure of A431 cells to a rapid temperature increase from 37° to 46°C could induce an increased expression (∼200% of control) and tyrosine phosphorylation/activation (∼300% of control) of protein kinase FA/glycogen synthase kinase-3α (kinase FA/GSK-3α) in a time-dependent manner, as demonstrated by an anti-kinase FA/GSK-3α immunoprecipitate kinase assay and by immunoblotting analysis with anti-kinase FA/GSK-3α and anti-phosphotyrosine antibodies. The heat induction on the increased expression of kinase FA/GSK-3α could be blocked by actinomycin D but not by genistein. In contrast, the heat induction on tyrosine phosphorylation/activation of kinase FA/GSK-3α could be blocked by genistein or protein tyrosine phosphatase, indicating that heat stress induces a dual control mechanism, namely, protein expression and subsequent tyrosine phosphorylation to cause cellular activation of kinase FA/GSK-3α. Taken together, the results provide initial evidence that kinase FA/GSK-3α represents a newly described heat stress–inducible protein subjected to tyrosine phosphorylation/activation, representing a new mode of signal transduction for the regulation of this human carcinoma dedifferentiation modulator and a new mode of heat induction on cascade activation of a protein kinase. J. Cell. Biochem. 66:16–26, 1997. © 1997 Wiley-Liss, Inc.     

Prevention of meningo/encephalomyelitis due to Sarcocystis neurona infection in mice is mediated by CD8 cells

Immunodeficient CD8 knockout mice were infected with Sarcocystis neurona merozoites, in order to determine the role of CD8 cells in protective immunity. Using a direct agglutination test, all infected mice seroconverted by selected time points. Infected mice developed splenomegaly and bilateral lymphadenopathy. Histological changes included marked follicular development in the spleen, endothelitis and moderate perivascular inflammation in the liver, and meningoencephalitis in the brain. Infected brains were positive for S. neurona by polymerase chain reaction. Corresponding to histopathological changes, there were decreased numbers of B-cells in the spleen. The mice did not have significant memory (CD44hi/CD4) or effector (CD45RBhi/CD4) populations present at the time of euthanasia. Flow cytometry confirmed the lack of CD8 cells. Taken together, these data support previous studies suggesting a critical role for CD8 cells in the prevention of menigoencephalitis in S. neurona-infected mice.     

Failure to upregulate cell surface PD-1 is associated with dysregulated stimulation of T cells by TGN1412-like CD28 superagonist

The CD28 superagonist (CD28SA) TGN1412 was administered to humans as an agent that can selectively activate and expand regulatory T cells but resulted in uncontrolled T cell activation accompanied by cytokine storm. The molecular mechanisms that underlie this uncontrolled T cell activation are unclear. Physiological activation of T cells leads to upregulation of not only activation molecules but also inhibitory receptors such as PD-1. We hypothesized that the uncontrolled activation of CD28SA-stimulated T cells is due to both the enhanced expression of activation molecules and the lack of or reduced inhibitory signals. In this study, we show that anti-CD3 antibody-stimulated human T cells undergo time-limited controlled DNA synthesis, proliferation and interleukin-2 secretion, accompanied by PD-1 expression. In contrast, CD28SA-activated T cells demonstrate uncontrolled activation parameters including enhanced expression of LFA-1 and CCR5 but fail to express PD-1 on the cell surface. We demonstrate the functional relevance of the lack of PD-1 mediated regulatory mechanism in CD28SA-stimulated T cells. Our findings provide a molecular explanation for the dysregulated activation of CD28SA-stimulated T cells and also highlight the potential for the use of differential expression of PD-1 as a biomarker of safety for T cell immunostimulatory biologics.     

Rapid accumulation of adoptively transferred CD8+ T cells at the tumor site is associated with long-term control of SV40 T antigen-induced tumors

We previously established a model to study CD8⁺ T cell (T_CD8)-based adoptive immunotherapy of cancer using line SV11 mice that develop choroid plexus tumors in the brain due to transgenic expression of Simian Virus 40 large T antigen (Tag). These mice are tolerant to the three dominant T_CD8-recognized Tag epitopes I, II/III and IV. However, adoptive transfer of spleen cells from naïve C57BL/6 (B6) mice prolongs SV11 survival following T_CD8 priming against the endogenous Tag epitope IV. In addition, survival of SV11 mice is dramatically increased following transfer of lymphocytes from Tag-immune B6 mice. In the current study, we compared the kinetics and magnitude of Tag-specific T_CD8 accumulation at the tumor site following adoptive transfer with a high dose of either Tag-immune or naïve donor cells or decreasing doses of Tag-immune lymphocytes. Following adoptive transfer of Tag-immune cells, epitope I- and IV-specific T_CD8 accumulated to high levels in the brain of SV11 mice, peaking at 5–7 days, while epitope IV-specific T_CD8 derived from naïve donors required three weeks to achieve peak levels. A similar delay in the peak of epitope IV-specific T_CD8 accumulation was observed when tenfold fewer Tag-immune donor cells were administered, reducing control of tumor progression. These results suggest that efficient and prolonged control of established autochthonous tumors is associated with high-level early accumulation of adoptively transferred T cells. We also provide evidence that although multiple specificities are represented in the Tag immune donor lymphocytes, epitope IV-specific donor T_CD8 play a predominant role in control of tumor growth.     

Key role of regulated upon activation normal T-cell expressed and secreted, nonstructural protein1 and myeloperoxidase in cytokine storm induced by influenza virus PR-8 (A/H1N1) infection in A549 bronchial epithelial cells

Influenza virus infection causes severe respiratory disease such as that due to avian influenza (H5N1). Influenza A viruses proliferate in human epithelial cells, which produce inflammatory cytokines/chemokines as a "cytokine storm" attenuated with the viral nonstructural protein 1 (NS1). Cytokine/chemokine production in A549 epithelial cells infected with influenza A/H1N1 virus (PR-8) or nonstructural protein 1 (NS1) plasmid was examined in vitro. Because tumor necrosis factor-α (TNF-α) and regulated upon activation normal T-cell expressed and secreted (RANTES) are predominantly produced from cells infected with PR-8 virus, the effects of mRNA knockdown of these cytokines were investigated. Small interfering (si)TNF-α down-regulated RANTES expression and secretion of RANTES, interleukin (IL)-8, and monocyte chemotactic protein-1 (MCP-1). In addition, siRANTES suppressed interferon (IFN)-γ expression and secretion of RANTES, IL-8, and MCP-1, suggesting that TNF-α stimulates production of RANTES, IL-8, MCP-1, and IFN-γ, and RANTES also increased IL-8, MCP-1, and IFN-γ. Furthermore, administration of TNF-α promoted increased secretion of RANTES, IL-8, and MCP-1. Administration of RANTES enhanced IL-6, IL-8, and MCP-1 production without PR-8 infection. These results strongly suggest that, as an initial step, TNF-α regulates RANTES production, followed by increase of IL-6, IL-8, and MCP-1 and IFNs concentrations. At a later stage, cells transfected with viral NS1 plasmid showed production of a large amount of IL-8 and MCP-1 in the presence of the H(2)O(2)-myeloperoxidse (MPO) system, suggesting that NS1 of PR-8 may induce a "cytokine storm" from epithelial cells in the presence of an H(2)O(2)-MPO system.     

Probing the infiltrating character of brain tumors: inhibition of RhoA/ROK-mediated CD44 cell surface shedding from glioma cells by the green tea catechin EGCg

Glioma cell-surface binding to hyaluronan (HA), a major constituent of the brain extracellular matrix (ECM) environment, is regulated through a complex membrane type-1 matrix metalloproteinase (MT1-MMP)/CD44/caveolin interaction that takes place at the leading edges of invading cells. In the present study, intracellular transduction pathways required for the HA-mediated recognition by infiltrating glioma cells in brain was investigated. We show that the overexpression of the GTPase RhoA up-regulated MT1-MMP expression and triggered CD44 shedding from the U-87 glioma cell surface. This potential implication in cerebral metastatic processes was also observed in cells overexpressing the full-length recombinant MT1-MMP, while the overexpression of a cytoplasmic domain truncated from of MT1-MMP failed to do so. This suggests that the cytoplasmic domain of MT1-MMP transduces intracellular signaling leading to RhoA-mediated CD44 shedding. Treatment of glioma cells with the Rho-kinase (ROK) inhibitor Y27632, or with EGCg, a green tea catechin with anti-MMP and anti-angiogenesis activities, antagonized both RhoA- and MT1-MMP-induced CD44 shedding. Conversely, overexpression of recombinant ROK stimulated CD44 release. Taken together, our results suggest that RhoA/ROK intracellular signaling regulates MT1-MMP-mediated CD44 recognition of HA. These molecular processes may partly explain the diffuse brain-infiltrating character of glioma cells within the surrounding parenchyma and thus be a target for new approaches to anti-tumor therapy.     

Expression of CEACAM1 or CEACAM5 in AZ‐521 cells restores the type IV secretion deficiency for translocation of CagA by Helicobacter pylori

Helicobacter pylori represents an important pathogen involved in diseases ranging from gastritis, peptic ulceration, to gastric malignancies. Prominent virulence factors comprise the vacuolating cytotoxin VacA and the cytotoxin‐associated genes pathogenicity island (cagPAI)‐encoded type IV secretion system (T4SS). The T4SS effector protein CagA can be translocated into AGS and other gastric epithelial cells followed by phosphorylation through c‐Src and c‐Abl tyrosin kinases to hijack signalling networks. The duodenal cell line AZ‐521 has been recently introduced as novel model system to investigate CagA delivery and phosphorylation in a VacA‐dependent fashion. In contrast, we discovered that AZ‐521 cells display a T4SS incompetence phenotype for CagA injection, which represents the first reported gastrointestinal cell line with a remarkable T4SS defect. We proposed that this deficiency may be due to an imbalanced coexpression of T4SS receptor integrin‐β₁ or carcinoembryonic antigen‐related cell adhesion molecules (CEACAMs), which were described recently as novel H. pylori receptors. We demonstrate that AZ‐521 cells readily express integrin‐β₁, but overexpression of integrin‐β₁ constructs did not restore the T4SS defect. We further show that AZ‐521 cells lack the expression of CEACAMs. We demonstrate that genetic introduction of either CEACAM1 or CEACAM5, but not CEACAM6, in AZ‐521 cells is sufficient to permit injection and phosphorylation of CagA by H. pylori to degrees observed in the AGS cell model. Expression of CEACAM1 or CEACAM5 in infected AZ‐521 cells was also accompanied by tyrosine dephosphorylation of the cytoskeletal proteins vinculin and cortactin, a hallmark of H. pylori‐infected AGS cells. Our results suggest the existence of an integrin‐β₁‐ and CEACAM1‐ or CEACAM5‐dependent T4SS delivery pathway for CagA, which is clearly independent of VacA. The presence of two essential host protein receptors during infection with H. pylori represents a unique feature in the bacterial T4SS world. Further detailed investigation of these T4SS functions will help to better understand infection strategies by bacterial pathogens.