Microbial cell responses to a non-ionic surfactant. II. Effects as assessed by fluorescein diacetate uptake

Summary The effects of a non-ionic surfactant, Pluronic F-68, on uptake of fluorescein diacetate (FDA) into yeast cells as measured by increase in fluorescence atca. 500 nm have been studied. The rate of FDA uptake was increased almost 2.5 fold by incubating cells with up to 5% commercial grade pluronic but this depended on source and degree of purity. Dye uptake was reduced by pre-purification of pluronic but this again depended on source of material. None of the pluronic preparations had any significant effects on the rate of enzymemediated FDA hydrolysis by cell-free extracts.     

Endocytosis of a monoclonal antibody recognising a cell surface glycoprotein antigen visualised using fluorescent conjugates

The endocytosis of a monoclonal antibody recognising a cell surface glycoprotein antigen has been investigated using several different fluorescent conjugates. These conjugates have been employed for both fluorescence microscopy to show the qualitative changes in distribution of antibody conjugates during endocytosis, and also flow cytofluorimetry to show the quantitative changes in fluorescence intensity associated with this redistribution. Using an antibody directly labelled with fluorescein it was difficult to demonstrate endocytosis due to the inability to distinguish clearly between internal and external fluorescence. However, a fluorescein-HSA-antibody conjugate which was heavily quenched at the cell surface was endocytosed and degraded during incubation at 37 degrees C for 4 h and was then visualised in a perinuclear distribution by the addition of agents to modify intracellular pH. This experiment demonstrated that such conjugates became localised within an acidic internal compartment. A tetramethylrhodamine-HSA-conjugate also demonstrated a similar perinuclear distribution but without the addition of endosomal pH modifiers. When used in conjunction with a fluorescein rabbit anti-HSA second label this conjugate also showed that not all conjugate was endocytosed during a 4-h period; some conjugate remained bound to the cell surface. These experiments suggested that endocytosis in this system differs from receptor-mediated endocytosis via coated pits which is reported to be more rapid and complete.     

Structure-based design of a bispecific receptor mimic that inhibits T cell responses to a superantigen

Key surface proteins of pathogens and their toxins bind to the host cell receptors in a manner that is quite different from the way the natural ligands bind to the same receptors and direct normal cellular responses. Here we describe a novel strategy for "non-antibody-based" pathogen countermeasure by targeting the very same "alternative mode of host receptor binding" that the pathogen proteins exploit to cause infection and disease. We have chosen the Staphylococcus enterotoxin B (SEB) superantigen as a model pathogen protein to illustrate the principle and application of our strategy. SEB bypasses the normal route of antigen processing by binding as an intact protein to the complex formed by the MHC class II receptor on the antigen-presenting cell and the T cell receptor. This alternative mode of binding causes massive IL-2 release and T cell proliferation. A normally processed antigen requires all the domains of the receptor complex for its binding, whereas SEB requires only the alpha1 subunit (DRalpha) of the MHC class II receptor and the variable beta subunit (TCRVbeta) of the T cell receptor. This prompted us to design a bispecific chimera, DRalpha-linker-TCRVbeta, that acts as a receptor mimic and prevents the interaction of SEB with its host cell receptors. We have adopted (GSTAPPA)(2) as the linker sequence because it supports synergistic binding of DRalpha and TCRVbeta to SEB and thereby makes DRalpha-(GSTAPPA)(2)-TCRVbeta as effective an SEB binder as the native MHC class II-T cell receptor complex. Finally, we show that DRalpha-(GSTAPPA)(2)-TCRVbeta inhibits SEB-induced IL-2 release and T cell proliferation at nanomolar concentrations.     

The mitogenic activity of staphylococcal enterotoxin B (SEB): a monovalent T cell mitogen that stimulates cytolytic T lymphocytes but cannot mediate their lytic interaction

Staphylococcal enterotoxin B (SEB), a monovalent T cell mitogen and inducer of T suppressor cells, was found to be a potent polyclonal activator of cytolytic T lymphocytes (CTL) effective against concanavalin A (Con A)-treated target cells. In addition to polyclonal stimulation of CTL, SEB could reactivate "memory" CTL, alloimmunized 60 to 90 days earlier, into "secondary" CTL detectable as early as 24 hr after onset of stimulation and specific for the original priming target cells. Optimal cytolytic activity was induced at 0.5 to 10 micrograms/ml SEB; optimal priming time was 3 days, correlating well with the proliferative activity and morphologic transformation of small lymphocytes into large T lymphoblasts. Long-term cultures of splenocytes, stimulated by SEB, continued to express high cytolytic activity. It is noteworthy that although SEB and Con A are comparable CTL inducers, SEB, unlike Con A, is an ineffective mediator of nonspecific, CTL/target cell interactions. To the best of our knowledge this is the first example of a CTL inducer unable to mediate CTL-target interaction and lysis. The latter observations suggests that different receptors are involved in CTL activation and in CTL-target interaction resulting in lysis.     

Zinc(II)-coordinated oligotyrosine: a new class of cell penetrating peptide

A new series of cell penetrating peptides (CPPs) are described. The peptides are oligomers of Tyr-ZnDPA, a tyrosine derivative with an appended 2,2'-dipicolylamine unit that forms a very stable coordination complex with a zinc (II) cation. This in turn allows reversible association with a chelating oxyanion such as a carboxylate or phosphate derivative. The peptide oligomers (Tyr-ZnDPA) n where n = 1, 2, 4, 8, are highly water soluble, but upon association with fatty acids or phospholipids they partition into an organic octanol phase. Furthermore, a fluorescent, fluorescein-labeled version of the octamer, (Tyr-ZnDPA) 8-Fl, can enter living mammalian cells via endocytosis and a biotin derivative can deliver fluorescein-labeled streptavidin. Fluorescence microscopy and flow cytometry experiments show that cell uptake is diminished by conditions that inhibit endocytosis. Additionally, uptake of (Tyr-ZnDPA) 8-Fl is greater than fluorescein labeled octaarginine (Arg 8-Fl) in all cell lines tested (CHO, COS-7, HeLa). Another difference with Arg 8-Fl is that cell uptake of (Tyr-ZnDPA) 8-Fl does not require the presence of heparan sulfate proteoglycans on the cell surface. This difference may eventually be of practical value because drug delivery systems that employ alternative endocytic mechanisms may be optimal for different cell lines or they may deliver selectively to different organelles within a cell.     

Ubiquitin E3 Ligase A20 is Required in Degradation of Microbial Superantigens in Vascular Endothelial Cells

The endothelial cells and tight junctions or adherens junctions form the endothelial barrier on the inner surface of the blood vessels. How the endothelial barrier degrades the endocytic microbial products, such as Staphylococcal enterotoxin B (SEB), is not fully understood yet. Ubiquitination is involved in protein degradation. This study aims to investigate the role of ubiquitin E3 ligase A20 (A20) in the degradation of endocytic SEB in endothelial cells. The human microvascular endothelial cell line, Hmvec, was cultured to monolayers in the inserts of transwells. SEB was added to the apical chambers to observe the endocytosis and degradation of SEB in Hmvecs. The fusion of endosome/lysosome was observed by immune staining. After exposed to SEB for 30 min, SEB was detected in Hmvecs. SEB could attach to the surface of Hmvecs and endocytosed into the cytoplasm of Hmvecs. The endocytosed SEB was degraded in the Hmvecs, which was transported to the transwell basal chambers in A20-deficient Hmvec monolayers. The SEB-carrying endosomes fused to the lysosomes in Hmvecs; the fusion of endosome/lysosome was disturbed in A20-deficient Hmvecs. In conclusion, A20 plays an important role in the degradation of the endocytic microbial product, SEB, in cardiac endothelial cells.     

Microbial cell responses to a non-ionic surfactant

Summary The effects of a non-ionic surfactant, Pluronic F-68, on growth of microbial cell cultures have been studied. Growth ofSaccharomyces cerevisiae at 30°C or 37°C as measured by viable cell counts was unaffected by culture with pluronic. However, corresponding absorbance measurements forS. cerevisiae incubated with 5–10% pluronic were lower than controls at both temperatures. Absorbance ofE. coli cultures was also significantly reduced by incubation with 5.0–10.0% pluronic at both temperatures although viable counts again revealed no significant inhibition of growth.     

Micelles of poly(oxyethylene)-poly(oxypropylene) block copolymer (pluronic) as a tool for low-molecular compound delivery into a cell: phosphorylation of intracellular proteins with micelle incorporated [gamma-32P]ATP.

Pluronic P85 (poly(oxyethylene)-poly(oxypropylene) block copolymer) was used for in vitro delivery of [gamma-32P]ATP into intact Jurkat cells. Negatively charged ATP molecules are not able to penetrate cell plasma membrane. Hence, exogenous [gamma-32P]ATP added to a cell culture does not participate in phosphorylation of intracellular proteins. The addition to cells of [gamma-32P]ATP solubilized in positively charged (containing dodecylamine) pluronic micelles results in considerable increase of protein phosphorylation. In this case the treatment of intact cells with alkaline phosphatase (resulting in dephosphorylation of external proteins) causes no essential decrease of [32P]-incorporation in cell proteins. That gives an evidence of delivery of solubilized ATP into a cell. Under the experimental conditions used, pluronic micelles neither influence the viability of cells nor permeabilize cell plasma membrane.     

Ubiquitin E3 ligase TNFIAP3 mediates endosome/lysosome fusion in nasal epithelial cells

The nasal epithelial barrier dysfunction is associated with the pathogenesis of nasal allergy; the causative factors are to be further elucidated. Ubiquitin E3 ligase TNFIAP3 (TNFIAP3, in short) plays a role in the maintenance of the homeostasis in the body. This study aims to elucidate the role of TNFIAP3 in the degradation of endocytic substances in nasal epithelial cells. The nasal epithelial cell line, RPMI 2650 cells (RPC), was cultured into monolayers in transwells. The endocytosis of staphylococcal enterotoxin B (SEB) by RPC monolayers was assessed by enzyme-linked immunoassay. The endocytosis of SEB-triggered endosome/lysosome fusion was observed by immunocytochemistry. The results showed that RPC monolayers expressed TNFIAP3 upon the endocytosis of SEB. Deficiency of TNFIAP3 resulted in abundant SEBs being transported to the basal chambers of transwells via the intracellular pathway. In the TNFIAP3-sufficient RPC, SEB-carrying endosomes fused with lysosomes were observed. The TNFIAP3-deficient RPC showed few SEB-carrying endosomes fused with lysosomes. In summary, TNFIAP3 plays an important role in tethering endosomes to lysosomes in RPC.     

Staphylococcus aureus enterotoxins A− and B: binding to the enterocyte brush border and uptake by perturbation of the apical endocytic membrane traffic

Enterotoxins of Staphylococcus aureus are among the most common causes of food poisoning. Acting as superantigens they intoxicate the organism by causing a massive uncontrolled T cell activation that ultimately may lead to toxic shock and death. In contrast to our detailed knowledge regarding their interaction with the immune system, little is known about how they penetrate the epithelial barrier to gain access to their targets. We therefore studied the uptake of two staphylococcal enterotoxins (SEs), SEA and SEB, using organ cultured porcine jejunal explants as model system. Attachment of both toxins to the villus surface was scarce and patchy compared with that of cholera toxin B (CTB). SEA and SEB also bound to microvillus membrane vesicles in vitro, but less efficiently than CTB, and the binding was sensitive to treatment with endoglycoceramidase II, indicating that a glycolipid, possibly digalactosylceramide, acts as cell surface receptor at the brush border. Both SEs partitioned poorly with detergent resistant membranes (DRMs) of the microvillus, suggesting a weak association with lipid raft microdomains. Where attachment occurred, cellular uptake of SEA and SEB was also observed. In enterocytes, constitutive apical endocytosis normally proceeds only to subapical early endosomes present in the actomyosin-rich “terminal web” region. But, like CTB, both SEA and SEB penetrated deep into the cytoplasm. In conclusion, the data show that after binding to the enterocyte brush border SEA and SEB perturb the apical membrane trafficking, enabling them to engage in transcytosis to reach their target cells in the subepithelial lamina propria.     

Activation-induced cell death in human T cells is a suicidal process regulated by cell density but superantigen induces T cell fratricide

Repeated ligation of the TCR results in apoptosis (activation-induced cell death; AICD). Superantigens such as Staphylococcal enterotoxin B (SEB) are particularly efficient at inducing AICD in T cells. We investigated whether apoptosis in human T cell subsets was due to fratricide (killing of neighboring cells) or suicide (cell autonomous death). AICD of Th1, Th2, Tc1, and Tc2 effector cells was dramatically enhanced at low cell densities and could be observed in single cell microcultures. AICD was unaffected by adhesion molecules or neighboring cells undergoing AICD, confirming the predominance of a suicidal mechanism. However, SEB was able to induce fratricidal apoptosis of type 1, but not type 2 cells. Fratricide was also observed when unstimulated T cells were exposed to activated Tc1 effector cells. Thus, AICD is tightly regulated to allow clonal T cell expansion and memory cell generation, but superantigens may subvert this process by allowing T cell fratricide.     

Endocytosis of wheat germ agglutinin binding sites from the cell surface into a tubular endosomal network

By using fluorescence and electron microscopy, the endocytic pathway encountered by cell surface components after they had bound wheat germ agglutinin (WGA) was visualized. The majority of these components are thought to consist of sialylated glycoproteins (HMWAG) that represent a subpopulation of the total cell surface proteins but most of the externally disposed plasma membrane proteins of the cell. Examination of semi-thin sections by medium- and high-voltage electron microscopy revealed the three-dimensional organization of vesicular and tubular endosomes. Binding of either fluorescein isothiocyanate-, horseradish peroxidase-, or ferritin-conjugated WGA to cells at 4 degrees C showed that the HMWAG were distributed uniformly over the cell surface. Warming of surface-labeled cells to 37 degrees C resulted in the endocytosis of WGA into peripheral endosomes via invagination of regions of both coated and uncoated membrane. The peripheral endosome appeared as isolated complexes comprising a vesicular element (300-400 nm diam.) surrounded by and continuous with tubular cisternae (45-60 nm diam.), which did not interconnect the endosomes. After 30 min or more label also became localized in a network of anastomosing tubules (45-60 nm diam.) that were located in the centrosomal region of the cell. Endocytosed WGA-HMWAG complexes did not become associated with cisternae of the Golgi apparatus, although tubular and vesicular endosomes were noted in the vicinity of the trans-Golgi region. The accumulation of WGA-HMWAG in the endosomes within the centrosomal region was inhibited when cells were incubated at 18 degrees C. None of these compartments contained acid phosphatase activity, a result that is consistent with other data that the HMWAG do not pass through lysosomes initially. The kinetics of labeling were consistent with the interpretation that recycling of most of the WGA binding surface glycoproteins occurred rapidly from early peripheral endosomes followed by the late trans-Golgi compartment. In conclusion, a portion of cell surface glycoproteins are routed to a complex arrangement of tubular and vesicular compartments following endocytosis that includes a putative post-endosomal, tubular reticulum that appears to be separate from the trans-most Golgi saccule.     

Influence of endocytosis-stimulating hormones on the protein binding capacity of the cell membrane

Histamine and serotonin enhanced the adsorption of fluorescein isothiocyanate (FITC)-labelled bovine serum albumin (BSA) to the membrane of Tetrahymena in a statistically significant and biologically relevant degree, while the similar action of insulin and epinephrine was not significant in either respect. The degree of hormonal influence on BSA-binding was similar to that exerted by the same hormones on the phagocytosis of protozoa in earlier studies. This supports the hypothetical implication that enhancement of protein adsorption to the cell membrane is an important element in the endocytosis stimulating action of histamine and serotonin.     

NPFXD-mediated endocytosis is required for polarity and function of a yeast cell wall stress sensor

The actin-associated protein Sla1p, through its SHD1 domain, acts as an adaptor for the NPFX(1,2)D endocytic targeting signal in yeast. Here we report that Wsc1p, a cell wall stress sensor, depends on this signal-adaptor pair for endocytosis. Mutation of NPFDD in Wsc1p or expression of Sla1p lacking SHD1 blocked Wsc1p internalization. By live cell imaging, endocytically defective Wsc1p was not concentrated at sites of endocytosis. Polarized distribution of Wsc1p to regions of cell growth was lost in the absence of endocytosis. Mutations in genes necessary for endosome to Golgi traffic caused redistribution of Wsc1p from the cell surface to internal compartments, indicative of recycling. Inhibition of Wsc1p endocytosis caused defects in polarized deposition of the cell wall and increased sensitivity to perturbation of cell wall synthesis. Our results reveal that the NPFX(1,2)D-Sla1p system is responsible for directing Wsc1p into an endocytosis and recycling pathway necessary to maintain yeast cell wall polarity. The dynamic localization of Wsc1p, a sensor of the extracellular wall in yeast, resembles polarized distribution of certain extracellular matrix-sensing integrins through endocytic recycling.     

T cell activation and retargeting using staphylococcal enterotoxin B and bispecific antibody: An effective in vivo antitumor strategy

 The aim of this work was to test for cure and immunity in a micrometastatic tumor model using in vivo T cell activation with staphylococcal enterotoxin B (SEB) and retargeting with antitumor×anti-CD3 F(ab′)₂ bispecific antibodies (bsAb). All studies were performed in C3H/HeN mice using syngeneic tumor cell lines. For survival studies, mice were injected intravenously on day 0 with CL62 (a p97-transfected clone of the K1735 murine melanoma tumor). Day-3 treatments included saline (control), SEB (50 γg intraperitoneal) with or without bsAb (5 μg i.v.). Cured mice, surviving beyond 60 days, were rechallenged with subcutaneous CL62, K1735, or a nonmelanoma control, AG104. SEB activation studies were performed with pulmonary tumor-infiltrating lymphocytes isolated from 10-day established CL62 tumors. Maximal tumor-infiltrating lymphocyte cytotoxicity was demonstrated 24 h following SEB injection, therefore bsAb treatments were administered 24 h after SEB. When survival was examined at 60 days, there were significantly more survivors in the group receiving SEB plus bsAb (70%) compared to the group receiving SEB alone (30%), and the controls (0%) (P=0.02 and P<0.01, respectively). Mice cured of CL62 using SEB alone or with bsAb demonstrated equal immunity to CL62, however, mice treated with SEB plus bsAb were more often immune to the p97^– parental cell line, K1735(P=0.001). Ag104 consistently grew in all mice. Results of these studies demonstrate that SEB plus bsAb can be effective, not only in curing tumors but also in providing protective immunity against targeted and nontargeted tumor antigens. Accepted: 14 October 1997     

Endocytosis of Xanthomonas campestris pathovar campestris lipopolysaccharides in non-host plant cells of Nicotiana tabacum

The specific recognition of phytopathogenic bacteria by plant cells is generally mediated by a number of signal molecules. The elicitor-active lipopolysaccharides (LPS) of the phytopathogenic bacterium Xanthomonas campestris pv. campestris (X.c.c) are recognized by its non-host plant Nicotiana tabacum (N.t.). This LPS was purified and labelled with fluorescein isothiocyanate (FITC) for monitoring the fate of these signal molecules in intact plant cells of tobacco. In this study we were able to show that the so-labelled LPS rapidly bound to the cell wall and was then internalized into the cells in a temperature- and energy-dependent way. This uptake of LPS could be outcompeted by the addition of an excess of unlabelled LPS. Furthermore, it was blocked by amantadine, an inhibitor of receptor-mediated endocytosis of mammalian cells. Immunolocalization experiments showed for the first time a significant co-localization of the LPS-elicitor with endosomal structures using an anti-Ara6 antibody. These observations suggest specific endocytosis of LPS(X.c.c.) into tobacco cells. The possibility for a receptor-mediated endocytosis comparable to the mammalian system will be discussed.     

Membrane translocation of penetratin and its derivatives in different cell lines

The third helix of the homeodomain of the Antennapedia homeoprotein can translocate through the cell membrane into the nucleus and can be used as an intracellular vehicle for the delivery of oligopeptides and oligonucleotides. A 16-amino acid-long peptide fragment, called penetratin, is internalized by the cells in a specific, non-receptor-mediated manner. For a better understanding of the mechanism of the transfer, penetratin and two analogs were synthesized:The conformation of penetratin peptides 1-3 was examined in both extracellular matrix-mimetic and membrane-mimetic environments. (1)H-NMR and CD spectroscopic measurements were performed in mixtures of TFE/water with different ratios. Peptides 1-3 were labeled by reacting their N-terminal free amino group with fluorescein isothiocyanate (FITC). Membrane translocation of the labelled peptides was studied with cell cultures [WEHI 164 murine fibrosarcoma cells (WC/1); chicken fibroblast cells (CEC-32); chicken monocytic cells (HD-11); human fibroblast (SV 80) and human monocytic cells (MonoMac-6)]. Confocal laser scanning microscopy and flow cytometry assay were used to study membrane translocation. Amphiphilicity was calculated for each peptide. In our experiments all the penetratin peptides penetrated into the cells. Helical conformation and membrane translocation ability showed little correlation: substitution of the two Trp with Phe increased the stability of helical conformation but decreased membrane translocation activity. The results of fluorescence microscopy and flow cytometry show that penetratin can be translocated into the cells by two mechanisms: endocytosis and direct transport through the cell membrane.     

Clathrin-independent endocytosis: a unique platform for cell signaling and PM remodeling

There is increasing interest in endocytosis that occurs independently of clathrin coats and the fates of membrane proteins internalized by this mechanism. The appearance of clathrin-independent endocytic and membrane recycling pathways seems to vary with different cell types and cargo molecules. In this review we focus on studies that have been performed using HeLa and COS cells as model systems for understanding this membrane trafficking system. These endosomal membranes contain signaling molecules including H-Ras, Rac1, Arf6 and Rab proteins, and a lipid environment rich in cholesterol and PIP(2) providing a unique platform for cell signaling. Furthermore, activation of some of these signaling molecules (H-Ras, Rac and Arf6) can switch the constitutive form of clathrin-independent endocytosis into a stimulated one, associated with PM ruffling and macropinocytosis.     

细胞内吞的研究进展

细胞外基质的各种分子经细胞膜进入真核细胞是一个复杂的过程。细胞内吞是通过细胞质膜的变形运动将细胞外物质转运入细胞内的过程。不同的细胞内吞途径需要不同的蛋白质分子参与,引起不同的信号转导通路。目前认为细胞内吞和膜转运是细胞对其信号转导过程的一种精密的组织安排,细胞内吞在细胞信号转导,维持机体动态平衡方面起着重要作用。细胞内吞途径通常可以分为网格蛋白依赖的内吞和非网格蛋白依赖的内吞,其中后者包括陷窝蛋白依赖和非陷窝蛋白依赖的内吞,以及巨胞饮介导的内吞。本文将就这几种主要细胞内吞途径及与细胞信号转导通路关系的研究进展予以介绍。     

Defective T cell receptor-mediated signal transduction in memory CD4 T lymphocytes exposed to superantigen or anti-T cell receptor antibodies

Lymphocytes must promote protective immune responses while still maintaining self-tolerance. Stimulation through the T cell receptor (TCR) can lead to distinct responses in naive and memory CD4 T cells. Whereas peptide antigen stimulates both naive and memory T cells, soluble anti-CD3 antibodies and bacterial superantigens stimulate only naive T cells to proliferate and secrete cytokines. Further, superantigens, like staphylococcal enterotoxin B (SEB), cause memory T cells to become anergic while soluble anti-CD3 does not. In the present report, we show that signal transduction through the TCR is impaired in memory cells exposed to either anti-CD3 or SEB. A block in signaling leads to impaired activation of the kinase ZAP-70 so that downstream signals and cell proliferation do not occur. We further show that the signaling defect is unique to each agent. In anti-CD3-treated memory T cells, the src kinase Lck is only transiently activated and does not phosphorylate and activate ZAP-70. In SEB-treated memory T cells, ZAP-70 does not interact with the TCR/CD3 complex to become accessible to Lck. Finally, we provide evidence that alternative signaling pathways are initiated in SEB-treated memory cells. Altered signaling, indicated by an elevation in activity of the src kinase Fyn, may be responsible for memory cell anergy caused by SEB. Thus, differentiation of naive T cells into memory cells is accompanied by alterations in TCR-mediated signaling that can promote heightened recall immunity or specific tolerance.