Cytocidal effects of Escherichia coli hemolysin on human T lymphocytes.

Escherichia coli hemolysin is the prototype of a large family of pore-forming toxins produced by gram-negative organisms. Besides its known cytotoxic activities against granulocytes, monocytes, endothelial cells, and renal epithelial cells, we now demonstrate that the toxin potently kills human T lymphocytes. Evidence based on different and independent approaches indicates that lymphocidal activity is due to formation of transmembrane pores. Additionally, cells prestimulated with phytohemagglutinin respond to low doses of E. coli hemolysin with DNA fragmentation similar to that observed in cells undergoing programmed cell death. Kinetic considerations lead us to conclude that DNA degradation may, however, represent an epiphenomenon. Killing of T cells is another means through which E. coli hemolysin could directly impair host defense.     

糖基化低密度脂蛋白对巨噬细胞及单核细胞的作用

我们观察了体外制备的非酶法糖基化低密度脂蛋白(glcLDL)在小鼠腹腔巨噬细胞(Mφ)及人单核细胞(U_(937)株)的降解代谢特点,发现小鼠腹腔Mφ在glcLDL的作用下未见胆固醇蓄积增加,对glcLDL的降解亦未见增加。U_(937)细胞对glcLDL的降解与glcLDL加样浓度成正比,用肉豆蔻酰乙酰佛波酯(TPA)处理细胞后其对glcLDL的降解较未处理者显著增加,且仍与加样浓度成正比。本文对上述现象与糖尿病时加速发生的动脉粥样硬化(AS)之间的关系作了初步分析。     

Some effects of lignocaine on cultured mouse peritoneal macrophages

Macrophages were exposed to lignocaine in perifused and non-perifused cultures using media with or without foetal calf serum. Effects on morphology, viability, phagocytosis and the release of enzymes were assessed. During the period of contact with lignocaine there was a selective release of -glucuronidase. After washing enzyme release continued over a period of 7 hours and, in the absence of foetal calf serum, a decrease in the total -glucuronidase content was found in non-perifused, cultures. Although lignocaine-treated cells phagocytosed particulates the rate of enzyme release was reduced compared with normal cells when subsequently exposed to quartz.     

Role of granulocytes and monocytes in experimental Escherichia coli endocarditis.

The role of granulocytes and monocytes during the induction and course of Escherichia coli endocarditis was investigated in rabbits by selectively depleting monocytes from the circulation with the drug VP16-213 and granulocytes and monocytes with nitrogen mustard. For induction, the number of E. coli needed to infect the vegetations in 50% of the rabbits was significantly lower in rabbits with combined granulocytopenia and monocytopenia than in those with selective monocytopenia or in control rabbits, whereas the number of E. coli needed to infect 50% of the rabbits did not differ between the latter two. During the course of the endocarditis in endocardial vegetations, the numbers of CFU per gram of vegetation were significantly higher in the rabbits with combined granulocytopenia and monocytopenia than in the monocytopenic and control rabbits but did not differ between the latter two. The numbers of granulocytes in the circulation and the numbers of CFU per gram of vegetation showed a significant negative correlation that was not measurably influenced by the duration of the disease but was dependent on the number of E. coli injected for the induction of endocarditis. Granulocytes were found to be most effective at the lowest numbers of bacteria injected. In the circulation, too, the numbers of CFU per milliliter were significantly higher in rabbits with combined granulocytopenia and monocytopenia than in those with selective monocytopenia and control rabbits, and there was a significant negative correlation between the numbers of granulocytes and CFU per milliliter of blood. From these findings we conclude that granulocytes play a protective role during the induction and course of E. coli endocarditis in rabbits, whereas no role is demonstrable for monocytes.     

Cytotoxic necrotizing factor type 1 of uropathogenic Escherichia coli kills cultured human uroepithelial 5637 cells by an apoptotic mechanism

Pathogenic Escherichia coli associated with urinary tract infections (UTIs) in otherwise healthy individuals frequently produce cytotoxic necrotizing factor type 1 (CNF1), a member of the family of bacterial toxins that target the Rho family of small GTP-binding proteins. To gain insight into the function of CNF1 in the development of E. coli-mediated UTIs, we examined the effects of CNF1 intoxication on a panel of human cell lines derived from physiologically relevant sites (bladder, ureters, and kidneys). We identified one uroepithelial cell line that exhibited a distinctly different CNF1 intoxication phenotype from the prototypic one of multinucleation without cell death that is seen when HEp-2 or other epithelial cells are treated with CNF1. The 5637 bladder cell line detached from the growth surface within 72 h of CNF1 intoxication, a finding that suggested frank cytotoxicity. To determine the basis for the unexpected toxic effect of CNF1 on 5637 cells, we compared the degree of toxin binding, actin fiber formation, and Rho modification with those CNF1-induced events in HEp-2 cells. We found no apparent difference in the amount of CNF1 bound to 5637 cells and HEp-2 cells. Moreover, CNF1 modified Rho, in vivo and in vitro, in both cell types. In contrast, one of the classic responses to CNF1 in HEp-2 and other epithelial cell lines, the formation of actin stress fibers, was markedly absent in 5637 cells. Indeed, actin stress fiber induction by CNF1 did not occur in any of the other human bladder cell lines that we tested (J82, SV-HUC-1, or T24). Furthermore, the appearance of lamellipodia and filopodia in 5637 cells suggested that CNF1 activated the Cdc42 and Rac proteins. Finally, apoptosis was observed in CNF1-intoxicated 5637 cells. If our results with 5637 cells reflect the interaction of CNF1 with the transitional uroepithelium in the human bladder, then CNF1 may be involved in the exfoliative process that occurs in that organ after infection with uropathogenic E. coli.     

Cytocidal and filamentous response of Escherichia coli cells exposed to low concentrations of hydrogen peroxide and hydroxyl radical scavengers

The hydroxyl radical scavengers thiourea, ethanol, and dimethyl sulfoxide significantly reduced the killing of Escherichia coli elicited by low concentrations of H2O2 (resulting in mode 1 killing) when treatments with the oxidant and the scavengers were performed in a complete growth medium (K medium) but not in M9 salts. In addition, thiourea efficiently prevented the toxic response to high concentrations of H2O2 (resulting in mode 2 killing) under both exposure conditions. Sod A cells, which do not respond with a bimodal pattern of toxicity when challenged with increasing concentrations of H2O2, were markedly protected by thiourea against the lethal action of various levels of the oxidant (which in the wild-type strain result in either mode 1 or mode 2 killing) under conditions of treatments in both K medium and M9 salts. In some experiments, wild-type cells were challenged with a low concentration of H2O2 (in the absence or presence of hydroxyl radical scavengers) and then postincubated in fresh K medium for various time intervals. It was found that scavengers were able to inhibit the filamentous response generated by exposure to the oxidant in K medium. Both the length and the number of filaments were markedly reduced. Treatment in M9 salts resulted in a limited number of very short filaments, and this response was slightly reduced by the scavengers.     

Immune modulatory effects of Prunella vulgaris L. on monocytes/macrophages

Prunella vulgaris L. (Labiatae), a popular Western and Chinese herbal medicine, has long been associated with anti-viral and anti-bacterial effects. While its anti-viral effects are attributed mainly to the inhibition of virus replication, the biological mechanisms of its anti-bacterial effects remain unknown. As a biological response modifier (BRM), the polysaccharides isolated from P. vulgaris have been shown to up-regulate the immune responses of monocytes/macrophages. However, the immune stimulatory effects seem to contradict its well-known anti-inflammatory properties. We hypothesized that the anti-microbial effects exhibited by the polysaccharides isolated from P. vulgaris encompass both anti-inflammatory and immune stimulatory effects. One of the polysaccharide fractions PV2IV markedly stimulated the production of superoxide and nitrite representing nitric oxide from murine macrophage RAW264.7 and brain macrophage BV2 cells. The amount of nitrite and superoxide produced after PV2IV stimulation was as high as that stimulated by bacterial endotoxin lipopolysaccharide (LPS) in a dose-dependent manner. In addition, PV2IV also increased cellular protein levels of inducible nitric oxide synthase (iNOS) and mRNA for tumor necrosis factor-alpha (TNFalpha). Similar to the effects of a high dose of LPS, the fraction PV2 could trigger activation-induced cell death (AICD) by stimulating caspase-3 activity and reduction of MTT uptake in monocytes/macrophages. These results may help our understanding of the molecular mechanism of P. vulgaris, which exhibited both immune stimulatory and anti-inflammatory effects against microbial invasion.     

Regulatory effects of IL-13 on synovial fluid macrophages and blood monocytes from patients with inflammatory arthritis.

Activated macrophages are central to the destructive processes of chronic inflammatory arthritis. In this study, it was hypothesized that IL-13, a product predominantly of 'Th2-type' lymphocytes, may be used therapeutically to down-regulate monocyte/macrophage activities at sites of chronic inflammation. Synovial fluid mononuclear cells were isolated from 12 patients with chronic inflammatory arthritis. Peripheral blood mononuclear cells (PBMC) were isolated at the same time as synovial fluid cells from all 12 patients. IL-13 significantly inhibited lipopolysaccharide (LPS)-induced tumour necrosis factor-alpha (TNF-alpha) production by mononuclear cells from peripheral blood, but not synovial fluid. In contrast, IL-13 inhibited LPS-induced IL-1 beta production by all cells, and as a positive response to IL-13, CD23 expression was increased on both cell populations. Blood monocytes cultured for 7 days with granulocyte-macrophage colony-stimulating factor (GM-CSF) or M-CSF responded to IL-13 in a manner similar to that detected for synovial fluid-derived cells, with suppression of LPS-induced IL-1 beta, but not TNF-alpha, production. In all experiments, the responses to IL-13 were very similar to those detected to IL-4, but differed from those measured with IL-10. Thus, the responses to IL-13 by synovial fluid cells and cultured monocytes are not equal to those of blood monocytes. The similar responses to IL-4 and IL-13 support claims of a common element for signalling from the IL-4 and IL-13 receptors. Furthermore, the activity of a common receptor chain may be altered by monocyte activation and differentiation.     

Pertussis toxin permeabilization enhances the traversal of Escherichia coli K1, macrophages, and monocytes in a cerebral endothelial barrier model in vitro

The occasionally severe neurological complications following the human respiratory tract infection 'whooping cough' have been attributed to pertussis toxin (PT) expressed by the causative agent Bordetella pertussis. Disruption of the endothelial blood-brain barrier (BBB) by PT might facilitate the translocation of immune cells and of hematogenous microbial pathogens. To test this hypothesis, we investigated whether PT enhances the traversal of bacteria employing human brain microvascular endothelial cells (HBMEC) as an in vitro endothelial barrier model. PT incubation significantly increased the translocation of Escherichia coli K1 across the HBMEC barrier. Only intercellular E. coli K1 bacteria could be identified by electron microscopy suggesting paracellular translocation. In addition, the migration of differentiated HL60-derived macrophages and of human monocytic U937 cells through PT-treated HBMEC barriers was also enhanced. In comparison to E. coli C600, E. coli K1 showed prolonged survival in translocated HL60-derived and J774 macrophages as well as in U937 monocytes which suggested a contribution of the 'Trojan horse' mechanism. In summary, our findings demonstrate that the PT-induced permeabilization of endothelial barriers enhances the paracellular transmigration of microbes and immune cells. In vivo, this activity might lower the threshold of bacteremia facilitating secondary cerebral infections and the subsequent development of brain pathologies.     

Studies of the immunological activities of the outer membrane protein from Escherichia coli

The outer membrane protein (OMP) prepared from Escherichia coli was found to be a potent mitogen for murine B cells and to be capable of inducing polyclonal antibody formation as well as a proliferative response. Spleen cells from nude mice responded equally as well to OMP as those from their normal litter-mates, whereas nylon-wool-purified T cells or thymocytes failed to respond. The proliferative response was dependent on the presence of macrophages. The macrophage dependency of the polyclonal antibody response seemed to be less than that of the proliferation. OMP was mitogenic for lipopolysaccharide (LPS)-resistant C3H/HeJ spleen cells, further indicating that OMP is an unique B-cell mitogen distinct from LPS.

OMP also enhanced the specific antibody response sixty-seven-fold to an optimal dose of sheep red blood cells (SRBC) in vitro. The kinetics of the response, however, was not altered from that of cultures without OMP. The anti-SRBC response of spleen cells from C3H/HeJ mice was also enhanced by the addition of OMP, suggesting that the adjuvant effects were not due to the LPS in the preparation. Antibody responses in vitro to TI-1 antigens, trinitrophenyl-LPS (Boivin) (TNP-LPS^B) and TNP-Brucella abortus, were not enhanced in the presence of OMP. In contrast OMP enhanced the response to TI-2 antigens, TNP-LPS^W (Westphal) and dinitrophenyl-Ficoll and T cells were shown to be required for these augmented antibody responses. Enhancement was not seen in nude mouse spleen cell cultures but was seen when nylon-wool-purified T cells were added to the cultures.

     

Effects of adhesins from mannose-resistant Escherichia coli on mediator release from human lymphocytes, monocytes, and basophils and from polymorphonuclear granulocytes.

We investigated the role of Escherichia coli expressing mannose-resistant hemagglutination and adhesins with regard to the induction of leukotrienes from a suspension of human lymphocytes, monocytes, and basophils (LMBs) compared with human polymorphonuclear granulocytes (PMNs). Genetically cloned E. coli strains expressing various types of mannose-resistant hemagglutination (MRH+) were phagocytosed to a higher degree by monocytes than the nonadherent E. coli strain. The various strains differed in their capacity to induce a chemiluminescence response, which showed the same pattern for LMBs and PMNs. Stimulation of LMBs with bacteria alone, unlike granulocytes, did not activate the cells for the release of leukotrienes. However, preincubation of LMBs with bacteria decreased subsequent leukotriene formation when the cells were stimulated with calcium ionophore. The inhibitory effect was dependent on the concentration of bacteria used for preincubation as well as on the preincubation temperature. The various bacterial strains differed in inhibitory potency for mediator release. Preincubation of LMBs with zymosan, opsonized zymosan, the bacterial peptide FMLP, and peptidoglycan had no inhibitory effect or even increased subsequent leukotriene formation. Opsonized bacteria were far less inhibitory than nonopsonized bacteria. In contrast to human LMBs, preincubation of human PMNs with mannose-resistant bacteria led to increased leukotriene B4 generation and reduced w-oxidation of leukotriene B4. Our data suggest that phagocytes (neutrophils, monocytes) respond in a different way for leukotriene formation after interaction with mannose-resistant E. coli.     

Expression of the VEP13 antigen (CD16) on native human alveolar macrophages and cultured blood monocytes

Human alveolar macrophages (AM phi) from thirteen patients, who were suffering from various lung diseases were harvested by bronchoalveolar lavage. Peripheral blood monocytes from eight healthy donors were isolated by Ficoll-Hypaque gradient centrifugation and adherence to plastic surface. To detect the VEP13 antigen (CD16) on these cells, a rosette assay employing ox erythrocytes coated by the CrCl3 method with purified VEP13 monoclonal antibody (Eo-VEP13) was used. A mean of 31.3% of freshly isolated AM phi and 3.9% of blood monocytes formed Eo-VEP13 rosettes. Monocytes cultured for 3 or 6 days in the presence of a supernatant from mouse L929 cells, which had been shown previously to improve long-term viability of human monocytes in culture, showed 12.5% and 25.3% Eo-VEP13 rosettes, respectively. No significant increase in VEP13 antigen expression was noted by culturing monocytes without L929 cell supernatant. The factor in L929 supernatant that induces VEP13 antigen expression has not been identified. Tunicamycin at 10 micrograms/ml inhibited significantly VEP13 antigen expression on monocytes. In contrast, IgG rosette formation was not reduced by tunicamycin. Our data show that subpopulations of native human AM phi and peripheral blood monocytes cultured in presence of a supernatant of L929 fibroblasts containing mainly murine CSF may express the CD16 antigen, which is normally found on large granular lymphocytes (LGL). Suppression by tunicamycin indicates that Fc receptor glycosylation takes place during a later differentiation step of mononuclear phagocytes.     

Cytokine profiling using monocytes/macrophages cultured on common biomaterials with a range of surface chemistries

Cytokines, chemokines, and growth factors were assayed from the supernatants of monocytes and macrophages cultured on common biomaterials with a range of surface chemistries. TNF-alpha, MCP-1, MIP-1alpha, IL-8, IL-6, IL-1beta, VEGF, IL-1ra, and IL-10 were measured from monocyte/macrophage cultures at different stages of activation and differentiation seeded onto polyethylene, polyurethane, expanded polytetrafluoroethylene, polymethyl methacrylate, and a hydrogel copolymer of 2-hydroxyethyl methacrylate, 1-vinyl-2-pyrrolidinone, and polyethylene glycol acrylate in tissue culture polystyrene (TCPS) plates. Empty TCPS wells and organo-tin polyvinyl chloride served as "blanks" and positive controls, respectively. Results showed an overall increase in cytokine, chemokine, and growth factor production as monocytes are activated or differentiated into macrophages and that proinflammatory and anti-wound healing cytokines and chemokines dominate this profile. However, cytokine production was only modestly affected by the surface chemistry of these four stable and noncytotoxic biomaterials.     

Activating effects of interferons, lymphokines and viruses on cultured chicken macrophages

Cultured chicken bone-marrow-derived and blood monocyte-derived macrophages could be activated by various treatments: (1) With crude lymphokine preparations obtained from Concanavalin A (ConA)-stimulated chicken spleen or thymus cell cultures: (2) with virus-induced interferons (IFN) from cultured chicken embryo fibroblasts or macrophages; (3) with inactivated reovirus or live infectious bursal disease virus (IBDV). Macrophage activity was expressed by cytostatic effects against lymphoblastoid target cells, and by morphological changes, such as enhanced spreading of the cells. The macrophage-activating capacity of lymphokine preparations (50% cytostasis-inducing dose) was closely associated with their antiviral activity (IFN units). According to its physico-chemical properties, ConA-induced lymphocyte interferon was considered to be IFN-gamma, but acid-and heat-resistant IFN-alpha or IFN-beta may also have been present in spleen cell or thymocyte culture supernatants. Virus-induced interferons (IFN-alpha/beta) were less effective in macrophage activation than in antiviral activity. Experimental results strongly suggested that macrophage activation by viruses was mediated by endogenous IFN.     

Entry and intracellular replication of Escherichia coli K1 in macrophages require expression of outer membrane protein A

Interactions between Escherichia coli K1, which causes meningitis in neonates, and macrophages have not been explored well. In this study we found that E. coli K1 was able to enter, survive, and replicate intracellularly in both murine and human macrophage cell lines, as well as in monocytes and macrophages of newborn rats. In addition, we demonstrated that OmpA (+) E. coli also enters and replicates in human peripheral blood monocytes in vitro. Outer membrane protein A (OmpA) expression on E. coli contributes to binding to macrophages, phagocytosis, and survival within macrophages. Opsonization with either complement proteins or antibody is not required for uptake and survival of the bacteria within the macrophages. Transmission electron microscopy and immunocytochemistry studies with the infected macrophages indicated that OmpA(+) E. coli multiplies enormously in a single phagosome and bursts the cell. Internalization of OmpA(+) E. coli by RAW 264.7 cells occurred by both actin- and microtubule-dependent processes, which are independent of RGD-mediated integrin receptors. Internalization and intracellular survival within phagocytic cells thus may play an important role in the development of bacteremia, which is crucial for E. coli crossing of the blood-brain barrier.     

Differential effects of cell adherence on LPS-stimulated cytokine production by human monocytes and macrophages

It is well known that adherence of monocytes (MO) to extracellular matrix substrates or tissue culture plastic activates these cells and induces the expression of a multitude of genes. Especially, it was described, that MO are primed by cell adhesion to produce higher amounts of some cytokines, e.g. interleukin (IL)-8 and tumor necrosis factor alpha (TNF-α). In order to investigate adherence-induced effects upon cytokine production, we seeded MO into tissue cultures and stimulated cells by lipopolysaccharide (LPS) simultaneously or at later time points. An increasing time-lag between cell adhesion and LPS-stimulation led to differential effects upon cytokine production: whereas TNF was upregulated (in accordance with reports by others), granulocyte colony-stimulating factor (G-CSF) was considerably down-regulated. In contrast, G-CSF production did not change, when cells were kept under non-adherent conditions in whole blood. In adherent cultures down-regulation of G-CSF could already be observed after two hours with a maximum after 24 h and was paralleled by a much lower abundance of G-CSF mRNA. Adhesion induced a significant suppression of G-CSF comparable to MO, if mature macrophages derived from MO in vitro were examined. Furthermore, two other cytokines, granulocyte-macrophage (GM)-CSF and IL-6, were also down-regulated following adhesion. In conclusion, activation of mononuclear phagocytes by adhesion can lead to -priming- for the production of some cytokines and at the same time to -silencing- for the production of others.     

Cytokine and growth factor production by monocytes/macrophages on protein preadsorbed polymers.

These studies evaluate the effect of biomedical polymers: Biomer, polydimethyl-siloxane (PDMS), polyethylene, expanded polytetrafluoroethylene (ePTFE), Dacron, and the control polystyrene with or without adsorbed proteins IgG, fibrinogen, and fibronectin on the ability of activated human monocytes/macrophages to produce Interleukin 1 Beta (IL-1-B), Interleukin 6 (IL-6), and Tumor Necrosis Factor Alpha (TNF-A). Monocytes/macrophages incubated on biomedical polymers with or without protein preadsorption produce variable levels of IL-1-B, IL-6, and TNF-A dependent on the polymer and adsorbed protein. IL-6 was produced in the greatest quantity and was the most influenced by protein adsorption. ePTFE and PDMS polymers were least stimulating while polystyrene was the most stimulating of monocyte activity. Adsorbed IgG consistently altered the ability of the polymers to activate monocytes/macrophages to produce cytokines. These studies provide important insight into conditions which modulate monocyte/macrophage activity in response to protein preadsorbed biomedical polymers.