Macrophage function in alloxan diabetic mice: expression of adhesion molecules,generation of monokines and oxygen and NO radicals

The increased incidence of bacterial and mycotic infections in poorly controlled diabetic patients or animals is frequently attributed to impaired activities of professional phagocytes (granulocytes, macrophages) in hypoinsulinaemic milieu. We measured production of monokines (IL-6 and tumour necrosis factor-alpha (TNF-α)), active NO and reactive oxygen intermediates (ROIs), as well as expression of several cell surface adhesion molecules (Mac-1, -2 and -3, intercellular adhesion molecule-1 (ICAM-1) and FcγRII), by thioglycollate medium-induced peritoneal macrophages of normoglycaemic and alloxan diabetic CBA/J mice (blood glucose level in the range 300 or 500 mg/dl). Macrophages of animals with moderate diabetes (300 mg/dl) produced significantly more IL-6 and TNF-α and ROIs than cells of control mice and showed an increased expression of all cell surface molecules, except Mac-3. NO/NO₂ production was not affected. Administration of insulin restored enhanced values to normal levels, except for the production of ROIs which remained unusually high. We conclude that two separate mechanisms influence macrophage physiology in diabetes—lack of saturation of insulin receptors on macrophages and an indirect effect due to formation of advanced glycosylation endproducts (AGE) on their surfaces. The latter is possibly responsible for increased generation of ROIs, since it cannot be down-regulated by prolonged insulin treatment. How the increased activity of macrophages of moderately diabetic mice (enhanced production of proinflammatory monokines and oxygen radicals as well as expression of molecules) is related to their ability to kill bacteria is now under investigation.     

Restricted expression of measles virus in primary rat astroglial cells

Persistent infection of the central nervous system (CNS) with measles virus (MV) is associated with characteristic restrictions of viral envelope gene expression as documented in subacute sclerosing panencephalitis (SSPE), measles inclusion body encephalitis (MIBE), or subacute measles encephalitis (SAME) in rats. To determine whether these restrictions are the result of a long lasting virus-host cell interaction or primarily based on intrinsic brain cell factors MV gene expression was analyzed in primary rat astroglial cultures. It could be shown that MV infection of these cells led to a defective replication cycle with a reduced synthesis of viral envelope proteins and a steep expression gradient of the monocistronic viral mRNAs similar to the findings in brain tissue of SSPE, MIBE, and SAME. This restriction of MV gene expression has not been observed in cells of nonneural origin. We suggest that this cell-type specific regulation of MV gene expression contributes to early events in the establishment of MV persistent infection in CNS tissue.     

Luminol-dependent chemiluminescence is related to the extracellularly released reactive oxygen intermediates in the case of rat neutrophils activated by formyl-methionyl-leucyl-phenylalanine

Luminol is a non-radical-specific amplifying molecule which produces light upon interaction with various reactive oxygen intermediates (ROIs). ROI production of rat peritoneal polymorphonuclear leukocytes (PMNLs) elicited by 2.3 microM formyl-methionyl-leucyl-phenylalanine (fMLP) results in a biphasic luminol-dependent chemiluminescence (LDCL) signal. Whereas ROIs are also produced intracellularly, as judged by flow cytometry, addition of non-membrane-permeable catalase reduces the first and second phases of the LDCL signal to around 3% and less than 3%, respectively. This suggests that in the case of fMLP-stimulated rat PMNLs, the LDCL signal is related to the ROIs in the extracellular medium and hydrogen peroxide has a key role in the formation of the LDCL signal. In the presence of the non-specific myeloperoxidase inhibitor Na-azide, the first phase of the LDCL signal decreases slightly (87+/-8%), while the second phase almost disappears (< 3%), indicating the myeloperoxidase dependence of the second phase. The hydroxyl radical scavenger histidine results in an 84+/-4% and a 71+/-4% decrease in the intensity of the first and second phases, respectively. Based on these data, it is concluded that hydrogen peroxide might be the source of hydroxyl radicals directly oxidizing luminol in the first phase of the LDCL signal, while in the second phase it serves as a substrate of myeloperoxidase in the peroxidation reaction of the luminol.     

Methyl-beta cyclodextrin alters the production and infectivity of Sendai virus

Cellular membrane cholesterol has been shown to support various membrane proteins. However, the role and function of membrane cholesterol in viral production are still unclear. Here, we investigated the effects of cholesterol depletion from the cell membrane on the production of hemagglutinating virus of Japan (HVJ; Sendai virus). Cholesterol depletion from LLC-MK2 cells by methyl-beta cyclodextrin treatment resulted in a marked increase in the production of both HVJ from the infected cells and virus-like particles from M-gene-transfected cells. The HVJ produced from cholesterol-depleted cells possessed a reduced amount of envelope cholesterol and showed a rather wide range of particle sizes and amount of envelope protein compared to HVJ produced from untreated cells. Direct depletion of envelope cholesterol from HVJ significantly impaired its infectivity, even without a change in envelope protein composition. These results suggest that membrane cholesterol plays important roles in regulating the production of infectious HVJ.     

Inhibition of avian sarcoma virus replication by glucosamine

The effect of glucosamine on the replication of avian sarcoma viruses has been studied. Six hours after addition of 20 μmoles/ml of glucosamine to the culture medium replication of infectious virus was inhibited by 99.9%. The production of viral particles detectable by physical techniques ceased in parallel with the synthesis of focus forming virus.Immunofluorescence studies indicate that the glucosamine block affects the production of viral envelope antigens. Accumulation of such antigens in the cytoplasm or at the cell surface was not observed in glucosamine-treated cultures, although the accumulation of a nonglycosylated polypeptide precursor cannot be excluded. Synthesis of group specific (gs1) protein was also inhibited by glucosamine, but to a lesser degree than that of viral envelope antigens.     

Receptors and ligands involved in viral induction of type I interferon production by plasmacytoid dendritic cells

Virus infection is sensed by the innate immune system which then rapidly initiates biosynthesis of type I interferon (IFN). The IFN signaling systems produce a broadly effective innate antiviral response by creating an antiviral state in both an autocrine and paracrine manner in cells and by activating innate and adaptive immunity. Plasmacytoid dendritic cells (pDCs) have the unique ability to produce very high levels of type I IFN following viral infection in vivo. Most recent research has focused on oligonucleotide-mediated induction of type I IFN production, implicating viral genome and replication intermediates as the stimulus for this response. However there are additional viral ligands which can potentially induce type I IFN production in pDCs, such as envelope glycoproteins, viral glycolipids, tegument, capsid or nuclear proteins. This area of viral immunology, which has been neglected in the literature, will be discussed here.     

CD4/CXCR4 co-expression allows productive HIV-1 infection in canine kidney MDCK cells

The Madin-Darby canine kidney (MDCK) cell line has become the prototypic cell type for studying the mechanisms involved in viral glycoproteins transport and viral assembly in polarized cells. This cell line has been used in our laboratories for studying human immunodeficiency virus (HIV-1), despite the fact that MDCK cells cannot be infected by HIV. In transfected MDCK cells, HIV-1 glycoproteins are specifically transported to the basolateral cell surface where viral budding also mostly occurs. However, this model is of limited use when viral propagation, infection of most cells, or larger production of virions, is needed. The initial objective of this work was thus to establish an MDCK-derived cell line that could be productively infected by HIV-1, in order to pursue our studies on the polarization of viral budding. Expression of both receptor and co-receptor for T-tropic strains of the virus showed that canine cells are rendered permissive once virus binding and entry is allowed. In addition, a reduced infectivity of the viral particles released from the basolateral surface was observed. This observation most likely reflects the interference mediated by CD4 molecules that accumulate at the basolateral domain. Accordingly, this effect was largely prevented when using viruses that down-regulate cell surface CD4 by expression of both viral accessory proteins Vpu and Nef. This is a further evidence that the function of different viral proteins depends of the site of viral budding, which is itself determined by the presence of targeting signal(s) harbored by viral envelope glycoproteins.     

Hypochlorous acid regulates neutrophil extracellular trap release in humans

Neutrophil extracellular traps (NETs) comprise extracellular chromatin and granule protein complexes that immobilize and kill bacteria. NET release represents a recently discovered, novel anti-microbial strategy regulated non-exclusively by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase generation of reactive oxygen intermediates (ROIs), particularly hydrogen peroxide. This study aimed to characterize the role of ROIs in the process of NET release and to identify the dominant ROI trigger. We employed various enzymes, inhibitors and ROIs to record their effect fluorometrically on in vitro NET release by human peripheral blood neutrophils. Treatment with exogenous superoxide dismutase (SOD) supported the established link between hydrogen peroxide and NET production. However, treatment with myeloperoxidase inhibitors and direct addition of hypochlorous acid (HOCl; generated in situ from sodium hypochlorite) established that HOCl was a necessary and sufficient ROI for NET release. This was confirmed by the ability of HOCl to stimulate NET release in chronic granulomatous disease (CGD) patient neutrophils which, due to the lack of a functional NADPH oxidase, also lack the capacity for NET release in response to classical stimuli. Moreover, the exogenous addition of taurine, abundantly present within the neutrophil cytosol, abrogated NET production stimulated by phorbol myristate acetate (PMA) and HOCl, providing a novel mode of cytoprotection by taurine against oxidative stress by taurine.     

Measurement of oxygen tension by the oxygen cathode

The usefulness of the flush-ended electrode forin vivo measurements of oxygen tension in tissue has been restricted by the lack of a satisfactory theoretical or empirical relationship between the polarographic current and the oxygen tension in the tissue. It is shown here that, if the electrode is small enough (so that its oxygen consumption is negligible compared with that of the nearby tissue), the current is proportional to the mean value over the electrode surface of the values that the oxygen tension would have taken if the potential had not been applied. The proportionality factor depends on the capillary-tissue structure in the neighbourhood of the probe. However, a membrane covering the surface of the electrode can be constructed so as to eliminate this effect and so enable comparisons between the levels of oxygen tension to be made at different points in the tissue. At the same time, the loss of sensitivity to variations of oxygen tension in time can be reduced to negligible proportions. The current, in this case, is proportional to the mean undisturbed value of the oxygen tension over the surface of the membrane.     

Involvement of reactive oxygen intermediates in tumor necrosis factor alpha-dependent bacteriostasis of Mycobacterium avium.

We studied the involvement of reactive oxygen intermediates and reactive nitrogen intermediates in the bacteriostasis of two Mycobacterium avium strains differing in virulence by resident peritoneal macrophages. We found that both the highly virulent strain (25291) and the low-virulence strain (1983) of M. avium induced superoxide production but inhibited nitrite production in vitro. This inhibition was due to the production of superoxide, a nitric oxide scavenger. The stimulation of superoxide production was two- to fivefold higher in strain 1983-infected than in strain 25291-infected resident peritoneal macrophages and was independent of contaminating T cells or NK cells. Superoxide secretion was dependent on the tumor necrosis factor (TNF) produced endogenously by the macrophages. This was also true when macrophages were isolated from infected mice. Addition of TNF to the infected resident peritoneal macrophages caused only a slight, albeit significant, increase in superoxide production by strain 25291-infected macrophages. Incubation of resident peritoneal macrophages with different scavengers of reactive oxygen intermediates showed that strain 1983 was susceptible to hydrogen peroxide produced by resident peritoneal macrophages. Strain 25291 was shown to decrease superoxide secretion inside heavily infected bone marrow-derived macrophages. This strain was also shown to be a better trigger for production of reactive oxygen intermediates than strain 1983. In summary, strain 1983 induced high levels of TNF synthesis that acted in an autocrine fashion to stimulate production of reactive oxygen intermediates by macrophages leading to growth restriction mediated by hydrogen peroxide. The highly virulent strain 25291 induced low levels of TNF synthesis, and therefore little reactive oxygen intermediate production, and could also inhibit superoxide production by the infected macrophages.     

Yeast-surface expressed BVDV E2 protein induces a Th1/Th2 response in naïve T cells

Yeast species such as Saccharomyces cerevisiae are known to be potent activators of the immune system. S. cerevisiae activates the innate immune system by engaging pattern recognition receptors such as toll like receptor 2 (TLR2) and dectin-1. In the current project, we express the immunogenic envelope protein E2 of bovine viral diarrhoea virus (BVDV) on the surface of S. cerevisiae. After successful expression, components of the innate and adaptive immune response induced by the recombinant S. cerevisiaein vitro were analysed to determine if expression in yeast enhances the immunogenicity of the viral protein. Recombinant S. cerevisiae stimulated production of the chemokine CXCL-8 in primary bovine macrophages, but did no stimulate production of reactive oxygen species (ROS) in the same cells. Additionally, bovine macrophages primed with S. cerevisiae expressing viral envelope proteins had a greater capacity for stimulating proliferation of CD4+ T-cells from BVDV-free animals compared to macrophages primed with envelope protein alone or S. cerevisiae without envelope protein expression. Heat inactivation of recombinant S. cerevisiae increased ROS production and capacity to stimulate CD4+ T-cells in macrophages but did not alter CXCL-8 release compared to the live counter-part. Additionally, heat-inactivation of recombinant S. cerevisiae induced less INFγ and IL-4 but equal amounts of IL-10 compared to live yeast T-cell cultures. Our studies demonstrate a use for S. cerevisiae as a vehicle for transporting BVDV vaccine antigen to antigen-presenting cell in order to elicit cell-mediated immunity even in na?ve animals.     

Monophosphoryl lipid A stimulated up-regulation of reactive oxygen intermediates in human monocytes in vitro

The production of reactive oxygen and nitrogen intermediates is a common response to infectious challenge in vivo. These agents have been implicated in the modulation of cytokine responses and are produced in large amounts in response to endotoxins produced by a number of infectious agents. The antigen-presenting cell activation caused by these lipopolysacchardies (LPS) has been exploited in the use of these agents as adjuvants. In recent years, less-toxic derivatives have been sought. One such agent, monophosphoryl lipid A (MPL), has been used increasingly in vivo as an adjuvant and as a modulator of the inflammatory process. It is known that this agent modulates the inflammatory response and cytokine production. In addition, we have shown its effect on the production of reactive nitrogen intermediates. In this paper, we show that MPL stimulates the release of high levels of superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)), the latter being greater than that seen with LPS and appearing to be related to the inability of MPL to stimulate catalase activity. When cells were pretreated with LPS or MPL and subsequently challenged with LPS, the production of O(2)(-) and H(2)O(2) was inhibited significantly by LPS and MPL. The concentration of MPL required to induce significant hyporesponsiveness to subsequent LPS challenge was 10 times lower than that of LPS. Hyporesponsiveness was greatest when induced by 10 microg/ml MPL, the same concentration that induced the maximum release of H(2)O(2) in primary stimulation. In addition, we have shown that following MPL pretreatment, LPS stimulation does not cause the loss of cytoplasmic IkappaBalpha, which occurs when human monocytes are cultured with LPS. From our results, we propose a model for the reduced toxicity of MPL.     

HIV gp120 V(1)/V(2) and C(2)-V(3) domains glycoprotein compatibility is required for viral replication

The envelope gene, especially the V(3) region, of HIV-1 has been shown to be a principal determinant of cell tropism, replication and cytopathogenicity of the virus. In addition, the V(1)/V(2) region of the envelope gene has been found to be an important factor in cell tropism. We examined the compatibility between the V(1)/V(2) and C(2)-V(3) domains of HIV-1 gp120 in different combinations on viral replication by using envelope recombinants between ME1 and ME46, two infectious molecular clones with diverse biologic activity longitudinally isolated from one seropositive subject. Our data demonstrate that a proper interaction between the regions of V(1)/V(2)and C(2) is essential for viral infection and hence replication. Sequence analysis and subsequent site directed mutagenesis study indicate that the pattern of potential envelope N-glycosylation in the V(1)/V(2) and C(2)-V(3) regions may be the determining factor in such interaction between these two regions. It is possible that improper N-glycosylation sites while not affecting virus assembly, can influence through steric hindrance the conformational change of the V(3) region that is required for the co-receptor attachment and hence the viral infectivity.     

The neutrophil-activating protein of Helicobacter pylori

Infection of the stomach mucosa by the gastric pathogen Helicobacter pylori is accompanied by a large infiltration of neutrophils and monocytes which are believed to contribute substantially to H. pylori-induced gastritis. A protein was identified (HP-NAP for neutrophil-activating protein from H. pylori) that was capable of increasing the adhesion of neutrophils to endothelial cells. We have demonstrated that HP-NAP is a dodecamer composed of identical 17-kDa subunits that induces the production of reactive oxygen radicals (ROIs) by neutrophils via a cascade of intracellular activation events. HP-NAP has also been shown to be chemotactic for neutrophils and monocytes, and a majority of H. pylori-infected patients have been found to produce antibodies specific for HP-NAP making it a strong vaccine candidate. More recently it has been shown that HP-NAP can stimulate tissue factor and plasminogen activator inhibitor-2 production by human monocytes. While structurally similar to the Escherichia coli DNA-binding protein Dps, HP-NAP has characteristics that are more similar to bacterioferritins being capable of binding up to 500 atoms of iron in vitro. Further study, however, has revealed that synthesis of HP-NAP in H. pylori is not altered by the addition or subtraction of metal ions from its growth medium suggesting that the primary role of the protein in vivo is not as a metal-binding protein. A number of other reports have proposed that HP-NAP acts as an adhesin being capable of binding several different compounds in vitro. Sequence analysis of the genomes of several other bacteria reveal that many possess Dps/HP-NAP-like proteins. The preliminary characterisation of some of these proteins will be discussed.     

Interferon-mediated inhibition of production of Gazdar murine sarcoma virus, a retrovirus lacking env proteins and containing an uncleaved gag precursor

HTG2 cells are murine sarcoma virus-transformed hamster cells. These cells continuously produce Gazdar sarcoma virus particles which are devoid of viral envelope proteins and which contain the uncleaved gag precursor polyprotein, Pr65, as their major protein constituent. Human interferon-alpha elicited an antiviral response in these cells as shown by the inhibition of replication of vesicular stomatitis virus in interferon-treated cells. Extracellular production of the retroviral particles by these cells was also inhibited by interferon in a dose-dependent manner and this inhibition was abolished by a specific antiserum to interferon. The intracellular level of Pr65 was not lowered in the interferon-treated cells, indicating that inhibition of viral protein synthesis was not responsible for inhibition of virus production. The present study suggests that interferon-mediated inhibition of retrovirus production, in general, is not a consequence of either a defective interaction between viral nucleoprotein cores and viral envelope proteins or a defect in the proteolytic processing of the gag polyprotein, since neither of these processes occurs during the morphogenesis of Gazdar particles and their production is nonetheless inhibited by interferon.     

Phenotypic mixing between murine oncoviruses and murine cytomegalovirus.

In vitro interactions between murine cytomegalovirus (MCMV) and murine leukaemia viruses (MuLV), two groups of enveloped viruses capable of causing persistent or latent infections in vivo, were examined for evidence of phenotypic mixing. The growth of MCMV in murine cells productively infected with ecotropic MuLV was shown to result regularly in the production of phenotypically mixed particles having the envelope antigens of MuLV and the genome of MCMV [MCMV(MuLV) pseudotypes]. The identity of such pseudotype particles was confirmed by the use of specific anti-MuLV serum and by the demonstration of restriction due to viral interference of penetration of these particles on MuLV-infected murine cells. This restriction was independent of N- or B-tropism. The production of reverse pseudotypes could not be examined because of the lytic effects of MCMV on the requisite assay cells.     

Induction of soluble antitumoral mediators by synthetic analogues of bacterial lipoprotein in bone marrow-derived macrophages from LPS-responder and -nonresponder mice

Macrophage-dependent antitumoral activity is partly mediated by soluble factors including cytokines, reactive-oxygen intermediates (ROIs), and reactive-nitrogen intermediates (RNIs). Activation of macrophages for tumor cytotoxicity can be achieved with various bacterial compounds, such as lipopolysaccharides (LPSs), muramyl-dipeptides, and lipopeptides. We studied the production and release of oxygen radicals, nitric oxide, and tumor necrosis factor alpha (TNF-alpha) by bone marrow-derived macrophages (BMDMs) of different mouse inbred strains after they were stimulated with the lipopeptide P3CSK4, a water-soluble synthetic analogue of the lipidated N terminus of bacterial lipoprotein. The lipopeptide was able to induce a strong, long lasting release of oxygen radicals in BALB/c mouse macrophages. Furthermore, it induced nitric oxide release from BMDMs of several mouse strains (BALB/c, C57Bl/6, C57Bl/10ScSn, Sv129, NMRI, and LPS-nonresponder C57Bl/10ScCr). Stimulation with P3CSK4 also resulted in comparable production of TNF-alpha in LPS-responder and nonresponder BMDMs from C57Bl/10ScSn mice and C57Bl/10ScCr mice, respectively. All three antitumoral mediators reached functional levels or concentrations as shown by the strong cytostatic/cytotoxic activity of lipopeptide-activated macrophages for the cell lines Abelson 8-1, M12.5/P815, and L929, which are sensitive to ROIs, nitric oxide, and TNF-alpha, respectively. We found that synthetic lipopeptides can induce the secretion of effective levels of soluble tumor-cytotoxic/cytostatic mediators in BMDMs of LPS-responsive and, of particular interest, also of LPS-unresponsive mice. This result could indicate that the highly effective bacterial-macrophage activators P3CSK4 and LPS use different receptors and/or different intracellular signal transduction pathways.     

The reversible formation of cysteine sulfenic acid promotes B-cell activation and proliferation

B-cell receptor (BCR) ligation generates reactive oxygen intermediates (ROIs) that play a role in cellular responses. Although ROIs can oxidize all macromolecules, it was unclear which modifications control B-cell responses. In this study, we demonstrate the importance of the first oxidation product of cysteine, sulfenic acid, and its reversible formation in B-cell activation. Upon BCR crosslinking, B cells increase ROI levels with maximal production occurring within 15 min. Increased ROIs preceded elevated cysteine sulfenic acid, which localized to the cytoplasm and nucleus. Analysis of individual proteins revealed that the protein tyrosine phosphatases (PTPs) SHP-1, SHP-2, and PTEN, as well as actin, were modified to sulfenic acid following BCR ligation. Additionally, we used 5,5-dimethyl-1,3-cyclohexanedione (dimedone), a compound that covalently reacts with sulfenic acid to prevent its further oxidation or reduction, to determine the role of reversible cysteine sulfenic acid formation in regulating B-cell responses. Dimedone incubation resulted in a concentration-dependent block in anti-IgM-induced cell division, accompanied by a failure to induce capacitative calcium entry (CCE), and maintain tyrosine phosphorylation. These studies illustrate that reversible cysteine sulfenic acid formation is a mechanism by which B cells modulate pathways critical for activation and proliferation.     

Enhanced macrophage anti-microbial activity following dimethylnitrosamine exposure in vivo is related to augmented production of reactive oxygen metabolites

Previous results demonstrated that mice exposed in vivo to DMN were more resistant to both bacterial and tumor challenges. Furthermore, macrophages (M phi) isolated from these animals demonstrated increased functional properties. As reactive oxygen intermediates (ROI) represent a key mechanism of anti-microbial action, it was important to determine whether ROI levels in M phi were related to augmented anti-microbial action in animals exposed to DMN in vivo. Peritoneal exudate M phi elicited with either thioglycollate (TG), Con A or C. parvum (CP) were examined for the production of ROIs. TG-M phi, Con A-M phi and CP-M phi obtained from animals exposed to DMN showed increased superoxide anion (O2-) production in vitro following stimulation with either phorbol myristate acetate (PMA) or opsonized zymosan (Op-zym) when compared to vehicle M phi. ROI production by bone marrow-derived macrophages (BMDM) produced by either GM-CSF or CSF-1 was also determined. BMDM from DMN-exposed animals obtained using either growth factor, had increased ROI production at 3, 5, 7 and 9 d of culture compared to vehicle BMDM. There was no shift in the kinetics of ROI production during differentiation of these BMDM. Analysis of extracellular anti-listericidal activity of TG- and CA-elicited M phi demonstrated that only TG-M phi obtained from DMN-exposed animals had enhanced killing capacity. There were no differences in intracellular anti-microbial activity in TG- and CA-elicited M phi obtained from either vehicle or DMN-exposed animals. TG-elicited M phi from either vehicle or DMN-exposed animals were examined for anti-microbial activity and H2O2 production following in vitro exposure to PMA. M phi from both vehicle and DMN treatment groups had enhanced killing and H2O2 production following PMA treatment, while PMA-stimulated TG-M phi from DMN-exposed animals demonstrated significantly higher levels of H2O2 production and cell killing as compared to all other treatment groups. These results suggest that previously observed increases in anti-microbial action by M phi from DMN-exposed animals are due in-part to enhanced ROI production.