Malaria circumsporozoite protein inhibits protein synthesis in mammalian cells.

Native Plasmodium circumsporozoite (CS) protein, translocated by sporozoites into the cytosol of host cells, as well as recombinant CS constructs introduced into the cytoplasm by liposome fusion or transient transfection, all lead to inhibition of protein synthesis in mammalian cells. The following findings suggest that this inhibition of translation is caused by a binding of the CS protein to ribosomes. (i) The distribution of native CS protein translocated by sporozoites into the cytoplasm as well as microinjected recombinant CS protein suggests association with ribosomes. (ii) Recombinant CS protein binds to RNase-sensitive sites on rough microsomes. (iii) Synthetic peptides representing the conserved regions I and II-plus of the P.falciparum CS protein displace recombinant CS protein from rough microsomes with dissociation constants in the nanomolar range. (iv) Synthetic peptides representing region I from the P.falciparum CS protein and region II-plus from the P.falciparum, P.berghei or P.vivax CS protein inhibit in vitro translation. We propose that Plasmodium manipulates hepatocyte protein synthesis to meet the requirements of a rapidly developing schizont. Since macrophages appear to be particularly sensitive to the presence of CS protein in the cytosol, inhibition of translation may represent a novel immune evasion mechanism of Plasmodium.     

Malaria sporozoites leave behind trails of circumsporozoite protein during gliding motility

As Plasmodium sporozoites undergo gliding motility in vitro, they leave behind trails of circumsporozoite (CS) protein that correspond to their patterns of movement. This light microscopic observation was made using Plasmodium berghei sporozoites, a monoclonal antibody (MAb H4) directed against the immunodominant repetitive epitope of the CS protein of P. berghei, and an immunogold-silver staining (IGSS) technique. Sporozoites pretreated with agents that inhibit sporozoite motility and invasiveness did not produce trails. Sporozoites that glided on microscope slides coated with MAb H4 left behind considerably longer CS protein trails than those on uncoated slides, and the staining of these trails was more intense. The fact that the CS protein is an exoantigen continuously released as trails by motile sporozoites, together with our previous finding that anti-CS protein antibodies inhibit sporozoite motility, strongly suggests that the CS protein plays a role in gliding motility. The sensitive IGSS technique used in this study may be a useful tool in the study of the translocation of surface proteins during gliding of other apicomplexans, other protists, and bacteria.     

Free zinc inhibits transport of vitamin C in differentiated HL-60 cells during respiratory burst

Zinc is an essential trace element for the immune system. It is known to be essential for highly proliferating cells, especially for cells of the immune system. However, zinc and other divalent cations are known to inhibit the human neutrophilic NADPH oxidase. Differentiated HL-60 cells were found to accumulate large quantities of vitamin C (ascorbate) after activation of the NADPH oxidase by phorbol esters (PMA). This increase in vitamin C transport is due to the generation of superoxide and subsequent oxidation of ascorbate to dehydroascorbate (DHA) which is preferentially taken up by the cells. We found that zinc reversibly inhibits both PMA-stimulated ascorbate uptake and superoxide generation with a half-maximal effect at 20 microM of free zinc ions. Higher residual extracellular ascorbate concentrations were measured with increasing zinc concentrations, indicating that less ascorbate was oxidized and taken up by the cells. When the fluorescent dye diSC3(5) was used to monitor shifts in membrane potential, we found that depolarization with PMA was prolonged after preincubation of the cells with zinc. Suppression of the respiratory burst as well as inhibition of the uptake of the antioxidant vitamin C may disturb the balance between oxidative damage of invading particles and antioxidant protection in activated neutrophils.     

Oxidative burst of Kupffer cells: target for liver injury treatment

Liver injury, and frequent consequent fibrosis, is the focus of a number of research groups ranging from molecular biologists to clinicians. It encompasses many aspects and approaches. Among biochemical events the role of Kupffer cells, oxidative stress and pro-inflammatory mediators are eminent. In this review we focus on recent findings into use of natural substances for the modulation of oxidative burst as well as production of inflammatory mediators by Kupffer cells.     

Induction of the respiratory burst in HL-60 cells. Correlation of function and protein expression

Differentiation of myeloid cells is associated with the gradual acquisition of functional capacity to produce a respiratory burst. In our study HL-60 cells were differentiated to the monocyte phenotype with IFN-gamma or 1,25-dihydroxyvitamin D3, or to the neutrophil phenotype with retinoic acid or DMSO to compare the time-course of expression of membrane and cytosolic oxidase components, and to correlate this with the appearance of a functional oxidase. Over a 6-day period of induction the rank order of the ability of these agents to induce expression of PMA-stimulated superoxide production was: IFN-gamma greater than 1,25(OH)2D3 greater than retinoic acid greater than DMSO. Immunoblot analysis of HL-60 membranes and cytosol was used to assess the amount of specific phagocyte oxidase factors (91 and 22 kDa subunits of membrane cytochrome b558 (gp91 and p22), and 47 and 67 kDa cytosol oxidase factors (p47 and p67)). HL-60 cell membranes or cytosol were tested in a cell-free assay of superoxide production by mixing with normal neutrophil cytosol or membranes, respectively. p47 was first detected at 16 h of differentiation, increasing similarly thereafter with all induction regimens and reaching a maximum by 3 to 4 days. The earliest detection of p67 varied from 2 to 6 days depending on the inducing agent and appeared to be the limiting cytosol component. Small amounts of both subunits of cytochrome b558 were detected in uninduced HL-60 membranes, but were sufficient to support substantial superoxide production when combined with normal neutrophil cytosol. Both cytochrome b558 subunit proteins and membrane oxidase activity increased during differentiation in parallel. We conclude that membrane and cytosol components of the NADPH oxidase complex appear at different times and increase differently during HL-60 differentiation. The production of p67 is the major factor limiting the respiratory burst during HL-60 differentiation.     

Effect of canine surfactant protein (SP-A) on the respiratory burst of phagocytic cells

Cells obtained from bronchoalveolar lavage, or neutrophils of peripheral blood of dog, were incubated with the canine surfactant-associated protein A (SP-A). A significant decrease of the production of Superoxide anion was observed after subsequent stimulation with phorbol-12-myristate-13-acetate (PMA) as measured by the lucigenin-dependent chemiluminesence (CL). Several other proteins used for control experiments did not decrease lucigenin-dependent CL, indicating a specific effect of SP-A on phagocytes. Treatment of SP-A with collagenase prior to incubation with neutrophils destroyed the depleting effect on oxygen radical production of PMA-stimulated cells. We propose that SP-A acts as a regulatory factor of the respitatory burst of alveolar macrophages and neutrophils in the lungs. The inhibitory effect of SP-A is down-regulated by collagenase released from stimulated alveolar macrophages.     

1-O-hexadecyl-2-Q-methylglycerol, a novel inhibitor of protein kinase C, inhibits the respiratory burst in human neutrophils

To assess the role of protein kinase C (Ca2+/phospholipid-dependent enzyme) in the activation of the human neutrophil respiratory burst, we have utilized an ether lipid of the type 1-O-alkyl-2-O-methylglycerol (AMG), recently shown to be an inhibitor of this kinase. AMG-C16 (with an hexadecyl chain at the sn-1 position) was found to inhibit the respiratory burst induced by sub-optimal concentrations of phorbol 12,13-dibutyrate. Respiratory burst activity was recovered by subsequent addition of a supraoptimal dose of phorbol 12-myristate 13-acetate, indicating that in the presence of the inhibitor only the activation of the NADPH:O2 oxidoreductase via protein kinase C is inhibited, but not the oxidoreductase itself. The respiratory burst induced by the chemoattractant N-formyl-methionyl-leucyl-phenylalanine (fMLP) was also inhibited in the presence of AMG-C16, the extent of inhibition being dependent on the concentration of fMLP. At the concentrations applied in these studies, AMG-C16 had no effect on cell viability, did not affect the formation of inositol phosphates induced by fMLP, and did not affect the characteristics of the Ca2+ fluxes induced by the same stimulus. In a cell-free assay system, AMG-C16 had no effect on the activity of cAMP-dependent or Ca2+/calmodulin-dependent protein kinase but inhibited protein kinase C in a dose-dependent fashion. To characterize the inhibitory action of AMG-C16 on the respiratory burst activity in more detail, we studied protein phosphorylation in relation to respiratory burst activity in neutrophil cytoplasts. We focused on the phosphorylation of the 47-kDa protein, because this protein is functionally associated with the NADPH:O2 oxidoreductase. At suboptimal concentrations of phorbol 12,13-dibutyrate, AMG-C16 inhibited phosphorylation of proteins, including that of the 47-kDa protein. Recovery of protein phosphorylation in parallel to recovery of respiratory burst activity was obtained by addition of increasing doses of phorbol 12,13-dibutyrate. Recovery of respiratory burst activity at intermediate concentrations of fMLP did not result in a proportional increase in 47-kDa protein phosphorylation; phosphorylation of the 47-kDa protein was recovered only at high concentrations of fMLP. From these data we conclude that protein kinase C is involved in the activation of the respiratory burst by phorbol esters and fMLP. However, with fMLP as a stimulus, a second signal seems to be triggered, which is insensitive to AMG-C16.     

Hydrocortisone inhibits the respiratory burst oxidase from human neutrophils in whole-cell and cell-free systems

The effects of hydrocortisone on the respiratory burst oxidase (NADPH oxidase, EC 1.6.99.6) from human neutrophils in both whole-cell and full soluble (cell-free) systems were investigated. In the whole-cell system, hydrocortisone inhibited the generation of superoxide by neutrophils exposed to phorbol myristate acetate, suggesting that steroids inhibit the bactericidal capacity of the body in an acute inflammatory phase. Hydrocortisone, which was added to the cuvette after the addition of NADPH and before the addition of sodium dodecyl sulfate, in a cell-free system, was found to inhibit the activation of superoxide-generating NADPH oxidase by sodium dodecyl sulfate. The concentration of hydrocortisone required for 50% inhibition of oxidase was 40 microM. Its inhibition was dose- and time-dependent in the cell-free system. However, hydrocortisone did not alter the Km of the oxidase for NADPH. These results suggest that steroids inhibit the reconstitution of NADPH oxidase by sodium dodecyl sulfate in the cell-free system, and that they do not alter the affinity to NADPH of the oxidase.     

Stimulators of AMP-activated protein kinase inhibit the respiratory burst in human neutrophils

In the present study, we have examined the potential ability of 5'-AMP-activated protein kinase (AMPK) to modulate NADPH oxidase activity in human neutrophils. AMPK activated with either 5'-aminoimidazole-4-carboxamide ribonucleoside (AICAR) or with 5'-AMP significantly attenuated both phorbol 12-myristate 13-acetate (PMA) and formyl methionyl leucyl phenylalanine-stimulated superoxide anion O2- release by human neutrophils, consistently with a reduced translocation to the cell membrane and phosphorylation of a cytosolic component of NADPH oxidase, namely p47phox. AMPK was found to be present in human neutrophils and to become phosphorylated in response to either AICAR or other stimulators of its enzyme activity. Furthermore, AICAR also strongly reduced PMA-dependent H2O2 release, and induced the phosphorylation of c-jun N-terminal kinase 1 (p46), p38 mitogen-activated protein kinase and extracellular signal-regulated kinase. Present data demonstrate for the first time that the activation of AMPK, in states of low cellular energy charge (such as under high levels of 5'-AMP) or other signals, could be a factor contributing to reduce the host defense mechanisms.     

Malaria sporozoites release circumsporozoite protein from their apical end and translocate it along their surface.

Plasmodium sporozoites, the causative agents of malaria, release circumsporozoite (CS) protein into medium when under conditions simulating those that the parasites encounter in the bloodstream of the vertebrate host. CS protein of the rodent parasite, Plasmodium berghei, is released as the lower molecular weight form, Pb44. This release is substratum- and antibody-independent. Previous studies show that CS protein is released at the trailing, posterior end of motile sporozoites. Video and electron microscopic studies now demonstrate that CS protein is released at the apical end of cytochalasin b-immobilized sporozoites. We propose that CS protein released from the apical end, the leading end of gliding sporozoites, adheres to the sporozoite surface and is translocated posteriorly by a cytochalasin-sensitive and apparently actin-mediated surface motor, which drives gliding motility. This model explains the mechanism of both the circumsporozoite precipitation (CSP) reaction and formation of the CS protein trail by gliding sporozoites.     

Vasoactive intestinal peptide inhibits the respiratory burst in human monocytes by a cyclic AMP-mediated mechanism

The neuropeptide vasoactive intestinal peptide (VIP) was shown to inhibit the production of reactive oxygen compounds (respiratory burst) in monocytes activated by serum opsonized zymosan. Reactive oxygen compounds are of importance for host defence against micro-organisms and cancer, but normal tissues are also susceptible to damage from these reactive substances. Maximum inhibition of respiratory burst was 40% by 0.1 microM VIP (ID100), while ID50 for the VIP effect was 0.36 nM VIP. PHM-27, closely related to VIP on the basis of the amino acid sequence, inhibited the respiratory burst with much lower potency (ID50 = 60 nM, ID100 = 1 microM). Secretin, related to VIP and PHM-27, produced no effect on the respiratory burst in monocytes. VIP was also shown to stimulate the cyclic AMP production in monocytes in a dose dependent manner. IBMX and forskolin, as well as the cyclic AMP analogue butyryl cyclic AMP were shown to produce an inhibition of the respiratory burst. In conclusion, this study showed that VIP inhibited the respiratory burst in monocytes by a cyclic AMP-mediated mechanism, and serves to establish still another role for VIP as a mediator in the neuro-immune axis.     

Ultrastructural localization of Plasmodium falciparum circumsporozoite protein in newly invaded hepatoma cells

The fate and disposition of the circumsporozoite (CS) protein of Plasmodium falciparum was investigated during hepatoma cell invasion with several sera raised against defined CS peptides, including both repeat and nonrepeat regions spanning approximately 60% of the P. falciparum CS gene product. Distribution of the protein, as revealed by immunoelectron microscopy, was limited to the surface of the sporozoite both before and after invasion. In particular, no CS protein antigen was detected in association with either the parasitophorous vacuole membrane or the host cell surface.     

Simulated microgravity impairs respiratory burst activity in human promyelocytic cells

Summary The concept of microgravity (free-fall) influencing cellular functions in nonadherent cells has not been a part of mainstream scientific thought. Utilizing rotating wall vessels (RWVs) to generate simulated microgravity conditions, we found that respiratory burst activity was significantly altered in nonadherent promyelocytic (HL-60) cells. Specifically, HL-60 cells in simulated microgravity for 6, 19, 42, 47, and 49 d had 3.8-fold fewer cells that were able to participate in respiratory burst activity than cells from 1×g cultures (P=0.0011, N=5). The quantity of respiratory burst products from the cells in simulated microgravity was also significantly reduced. The fold increase over controls in mean fluorescence intensities for oxidative products from cells in microgravity was 1.1±0.1 versus 1.8±0.3 for cells at 1 ×g (P=0.013, N=4). Furthermore, the kinetic response for phorbol ester-stimulated burst activity was affected by simulated microgravity. These results demonstrate that simulated microgravity alters an innate cellular function (burst activity). If respiratory burst activity is impaired by true microgravity, then recovery from infections during spaceflight could be delayed. Finally, RWVs provide an excellent model for investigating the mechanisms associated with microgravity-induced changes in nonadherent cells.     

Ivermectin inhibits activation of Kupffer cells induced by lipopolysaccharide toxin]

Lipopolysaccharide-stimulated liver macrophages (Kupffer cells) secrete many physiologically active substances responsible for inflammatory reaction of the organism. The mechanism by which ivermectin, a macrocyclic lactone possessing a broad antiparasitic activity, modulates basic effects elicited by lipopolysaccharide in the primary culture of rat Kupffer cells was studied. It was found that ivermectin in the absence of endotoxin did not affect a functional state of the Kupffer cells. Preincubation of Kupffer cells with ivermectin (1 mM), however, significantly blocked response to the subsequent administration of lipopolysaccharide (1 mg/ml). In particular, secretion of tumor necrosis factor TNF alpha, nitric oxide NO and prostaglandin E2 was suppressed. Also, an LPS-triggered rise in the intracellular concentration of calcium ions was less pronounced. Removal of chloride anions from the extracellular medium completely abolished inhibitory effects of ivermectin. It is suggested that invermectin exerts its action via binding to the glycine-gated chloride receptors/channels of the Kupffer cells, which may reduce toxic reactions manifestations observed under infections caused by Gram-negative bacteria.     

Hypoxia selectively inhibits respiratory burst activity and killing of Staphylococcus aureus in human neutrophils

Neutrophils play a central role in the innate immune response and a critical role in bacterial killing. Most studies of neutrophil function have been conducted under conditions of ambient oxygen, but inflamed sites where neutrophils operate may be extremely hypoxic. Previous studies indicate that neutrophils sense and respond to hypoxia via the ubiquitous prolyl hydroxylase/hypoxia-inducible factor pathway and that this can signal for enhanced survival. In the current study, human neutrophils were shown to upregulate hypoxia-inducible factor (HIF)-1α-dependent gene expression under hypoxic incubation conditions (3 kPa), with a consequent substantial delay in the onset of apoptosis. Despite this, polarization and chemotactic responsiveness to IL-8 and fMLP were entirely unaffected by hypoxia. Similarly, hypoxia did not diminish the ability of neutrophils to phagocytose serum-opsonized heat-killed streptococci. Of the secretory functions examined, IL-8 generation was preserved and elastase release was enhanced by hypoxia. Hypoxia did, however, cause a major reduction in respiratory burst activity induced both by the soluble agonist fMLP and by ingestion of opsonized zymosan, without affecting expression of the NADPH oxidase subunits. Critically, this reduction in respiratory burst activity under hypoxia was associated with a significant defect in the killing of Staphylococcus aureus. In contrast, killing of Escherichia coli, which is predominantly oxidase independent, was fully preserved under hypoxia. In conclusion, these studies suggest that although the NADPH oxidase-dependent bacterial killing mechanism may be compromised by hypoxia, neutrophils overall appear extremely well adapted to operate successfully under severely hypoxic conditions.     

Mechanism of inhibition by cyclic AMP of protein kinase C-triggered respiratory burst in Ehrlich ascites tumour cells

The superoxide anion generation in Ehrlicg ascites tumour (EAT) cells increased more than two-fold in the presence of the tumour promoter, tetradecanoyl phorbol myristate acetate (TPA). Epinephrine and dibutryl cAMP (Bt₂ cAMP) inhibited in a dose-dependent manner, both basal and TPA-triggered superoxide generation in EAT cells. The kinetics of inhibition of superoxide generation showed a maximum inhibition between 30 and 40 min of preincubation with epinephrine or Bt₂ cAMP of EAT cells and coincided with an increase in activity of a phosphoprotein phosphatase. In TPA-treated EAT cells, epinephrine or Bt₂ cAMP increased the phosphatase activity in a dose-dependent manner. In vitro EGTA, EDTA and sodium fluoride inhibited phosphatase activity. Superoxide generation in response to TPA in Triton-permeabilized EAT cells was inhibited by inclusion of the phosphatase in the assay. Taken together, these results clearly suggest that the phosphatase activity in EAT cells develops as a result of protein kinase A (PKA) and protein kinase C (PKC)-mediated phosphorylation of the phosphatase which then mediates dephosphorylation of the PKC-triggered phosphorylation of proteins to inhibit respiratory burst. A cross-talk between PKA and PKC pathways negatively modulates superoxide generation in EAT cells.     

Turning on the respiratory burst

The respiratory burst is a distinguishing property of phagocytes. It is induced by chemotactic stimulation or phagocytosis and reflects the activation of a membrane-bound enzyme system that transfers electrons from cytosolic NADPH to extracellular oxygen, producing superoxide. The products of the burst are essential for the killing of microorganisms, but are also a cause of tissue damage and inflammation. Studies aimed at a better understanding of the regulation of the respiratory burst should help in the search for new ways to treat infections and inflammation.     

Activity of plant extracts on the respiratory burst and the stress protein synthesis

Aqueous, methanol and dichloromethane extracts from Artemisia copa, Baccharis grisebachii, Baccharis incarum, Baccharis latifolia, Mutisia kurtzii and Pluchea sagittalis, plants used in the Traditional Medicine of South America, are studied for activity on the respiratory burst and the inducible heat shock protein of 72 kD (hsp72) synthesis. Activity on the production of reactive oxygen species (ROS) and reactive nitrogen species (RNS), as well as on hsp72 synthesis was measured by flow cytometry in human neutrophils. Cells were stimulated using hydrogen peroxide, phorbol-12-myristate-13-acetate (PMA) or formyl-methionyl-leucyl-phenylalanine (FMLP) for ROS generation, and sodium nitroprusside (SNP) or PMA in the presence of calmodulin inhibitor W-13 for RNS. The production of hsp72 was induced by heat, PMA, H2O2 and SNP. The best inhibitory activity was shown by the dichloromethane extracts of Baccharis grisebachii and Pluchea sagittalis that were active in all the assays. The aqueous extract of Pluchea sagittalis was also active in most assays. The aqueous extract from Mutisia kurtzii caused a clear increase of the hsp72 production and showed prooxidant activity.     

On the evolutionary history of the circumsporozoite protein in plasmodia

We report the complete nucleotide sequence of the circumsporozoite (CS) gene of Plasmodium brasilianum and present an analysis of its evolutionary profile. Despite the lack of a reliable time scale, the analysis of the number and distribution of fixations among seven taxa provides a first glimpse of the evolutionary history of the CS gene, and suggests that the branching events of this gene are completely unconnected with--and far precede in time--the speciation event of the parasite's vertebrate hosts.