P2Z adenosine triphosphate receptor activity in cultured human monocyte- derived macrophages

The present study shows that human mononuclear phagocytes express a P2Z- like purinergic membrane receptor activity. Extracellular adenosine triphosphate (ATP) induces the formation of nonselective membrane pores in human mononuclear phagocytes that allow the entry of otherwise membrane impermeant fluorescent dyes (YO-PRO-1 or Lucifer yellow) into the cytoplasm of these cells. The percentage of mononuclear phagocytes that was permeabilized by ATP increased as monocytes matured into macrophages. Their response to ATP was inhibited by Mg2+ and oxidized ATP. Benzoylbenzoic-ATP (BzBzATP) was approximately 60% as effective as ATP and adenosine-5 -O-(thiophosphate) (ATP gamma S) was less than 20% as effective as ATP in permeabilizing human macrophages to YO-PRO-1 or Lucifer Yellow. Thus, the human P2Z-like receptor differs from its murine counterpart because BzBzATP, ATP, and ATP gamma S are equally efficacious in permeabilizing murine macrophage-like J774 cells to these dyes. UTP, GTP, and CTP were ineffective in permeabilizing human or murine macrophages to YO-PRO-1. Taken together, these data indicate that human monocyte-derived macrophages express a P2Z-like activity that is pharmacologically distinct from that expressed by their murine counterparts and that expression of these receptors is developmentally regulated in human mononuclear phagocytes.     

Degradation of tissue-type plasminogen activator by human monocyte- derived macrophages is mediated by the mannose receptor and by the low- density lipoprotein receptor-related protein

The balance of tissue-type plasminogen activator (t-PA) production and degradation determines its concentration in blood and tissues. Disturbance of this balance may result in either increased or decreased proteolysis. In the present study, we identified the receptor systems involved in the degradation of t-PA by human monocytes/macrophages in culture. Monocytes were cultured and became macrophages within 2 days. At 4 degrees C, 125I-t-PA bound to macrophages with high (apparent dissociation constant [kd], 1 to 5 nmol/L) and low affinity (kd > 350 nmol/L). At 37 degrees C, the cells internalized and degraded t-PA via the high affinity binding sites, which were partially inhibited by mannan. The low affinity binding sites were 6-aminohexanoic acid- inhibitable and not involved in t-PA degradation. Degradation of t-PA was upregulated during differentiation of monocytes to macrophages. Dexamethasone further upregulated the mannan-inhibitable t-PA degradation. Lipopolysaccharide downregulated both mannan-inhibitable and non-mannan-inhibitable t-PA degradation. Non-mannan-inhibitable degradation was completely blocked by recombinant 39-kD receptor- associated protein (RAP, inhibitor of lipoprotein receptor-related protein [LRP]), whereas mannan-inhibitable degradation was blocked by the addition of a monoclonal antibody against the mannose receptor. No differences between the degradation of t-PA and functionally inactivated t-PA were observed. We conclude that human monocyte-derived macrophages are able to bind, internalize, and degrade t-PA. Degradation of t-PA does not require complex formation with plasminogen activator inhibitors. The macrophages use two independently regulated receptors, namely, the mannose receptor and LRP, for the uptake and degradation of t-PA.     

Chlamydia pneumoniae infection of alveolar macrophages: a model

Chlamydia (Chlamydophila) pneumoniae is a common respiratory pathogen, and it seems likely that alveolar macrophages may have an important role in infection with this bacterium. In the present study, we examined the usefulness of a continuous cell line of murine alveolar macrophages, designated "MH-S," as an in vitro C. pneumoniae infection model. Infection of MH-S cells with C. pneumoniae resulted in the development of typical inclusion bodies in the cells, similar to that seen in primary alveolar macrophages. However, we noted that, although the number of bacteria in the cultures increased during the infection, there was a restricted production of infective elementary bodies. The analysis of bacterial messenger RNA in the cultures showed that the message levels for the omcB gene were present only at a moderate level, but the levels of hsp60 messages increased markedly during infection. Neutralization of tumor necrosis factor (TNF)-alpha induced by inoculation with antibody significantly enhanced the infection, but omcB message levels were still inhibited. These results indicate that the growth of C. pneumoniae in alveolar macrophages may be restricted. Endogenous TNF-alpha may be one of the factors responsible for such restriction, but other factors also may be involved.     

Interactions of Chlamydia pneumoniae with human endothelial cells

In order to fulfill the "biological plausibility" criterion of a role for infection with Chlamydia pneumoniae in the pathogenesis of human atherosclerosis, detailed studies on the interaction of this organism with the cell types involved are necessary. This article summarizes the current knowledge on the interaction of C. pneumoniae with human endothelial cells. In vitro, C. pneumoniae can infect human endothelial cells and induce the expression of many molecules that are important mediators of atherogenesis including cytokines, adhesion molecules, chemokines, and molecules with procoagulant activity.     

Antibody response to the 60-kDa heat-shock protein of Chlamydia pneumoniae in patients with coronary artery disease

Serum specimens from 752 individuals undergoing coronary arteriography were examined for antibodies to Chlamydia pneumoniae. Patients with coronary artery disease (CAD) were more likely to have IgG antibodies to C. pneumoniae than were individuals without CAD (60% vs. 52%; P=.007; odds ratio, 1.8; 95% confidence interval, 1. 17-2.77). Antibodies to recombinant hsp60 of C. pneumoniae were found with nearly the same frequency in patients with CAD and individuals without CAD (29% vs. 30%; P=.751). There was no association between chlamydial hsp60 antibodies and the severity of CAD or a previous myocardial infarction. Patient sera reacted most frequently to C. pneumoniae proteins of 17, 38, 40, 58, and 60/62 kDa. Reactivity to these proteins was not different between patients with and without CAD. Study results indicate that neither antibodies to chlamydial hsp60 nor antibodies to other C. pneumoniae proteins are useful for discriminating between seropositive patients with and without CAD.     

Potential relevance of Chlamydia pneumoniae surface proteins to an effective vaccine

The surface of Chlamydia pneumoniae is covered with proteins but their exact identification is not known probably because of the presence of conformational epitopes. A family of 21 pmp genes has been found by DNA sequencing. In common, these genes have the capacity to encode the amino acid motif GGAI. Several of the genes have the capacity to encode outer membrane proteins of about 100 kDa. Thus, they are candidate genes to encode the protein(s) present in the 98-kDa protein band of the C. pneumoniae outer membrane complex. The production of recombinant GGAI proteins is described as is the use of polyclonal antibodies raised against the recombinant GGAI proteins to determine their expression in C. pneumoniae elementary bodies. At least three of the proteins, Omp4, 5, and 11, are expressed.     

Killing of Plasmodium falciparum by human monocyte–derived macrophages

Freshly isolated human peripheral blood monocytes inhibited the growth of blood-stage asexual Plasmodium falciparum parasites in vitro. The monocytes contained intracellular parasite pigment and a few whole parasites, but the remaining parasites reinvaded fresh red cells successfully and were morphologically normal. Anti-parasitic activity of these macrophages was not significantly enhanced by treatment with recombinant tumour necrosis factor alpha, recombinant gamma-interferon or lymphoblastoid alpha-interferon. Catalase had no effect on this parasite inhibition, suggesting a hydrogen peroxide independent mechanism. Anti-parasitic activity was, however, enhanced by prior maturation of the monocytes. Monocytes matured for 6 days caused 100% killing of parasites. In contrast to identical concentrations of freshly isolated monocytes the parasites incubated with these matured macrophages showed intraerythrocytic death similar to the crisis forms seen in vivo. gamma-interferon present either during the assay or as a pretreatment had no significant enhancing effect on the killing, although cytotoxicity to tumour cell lines was enhanced. Conditioned medium from macrophages showed only moderate parasite inhibition.     

Cigarette smoking decreases interleukin-8 secretion by human alveolar macrophages

Cigarette smoking can impair pulmonary immune function, and hence influences the development of lung diseases. Interleukin-8 (IL-8) is a proinflammatory peptide and a potent chemotactic factor for neutrophils, and is produced by both immune and non-immune cells including monocytes and alveolar macrophages (AM). We investigated the effect of cigarette smoking on the secretion of IL-8 by human AM. The IL-8 concentration in bronchoalveolar lavage fluid (BALF) was much higher in smokers than in non-smokers (18.4 +/- 3.9 vs 4.1 +/- 1.0 pg ml-1; P < 0.005). However, spontaneous IL-8 secretion by cultured AM was lower in smokers than in non-smokers (46.8 +/- 12.7 vs 124.1 +/- 24.0 ng ml-1; P < 0.01). When stimulated with lipopolysaccharide (LPS), AM from smokers secreted significantly less IL-8 than those from non-smokers at all tested concentrations of LPS. In contrast, the amount of IL-8 secreted by peripheral blood monocytes with or without LPS stimulation was comparable in smokers and non-smokers. These observations indicate that smoking decreases IL-8 secretion by AM, which may modify or decrease the inflammatory response in the lung.     

Evidence for persistent Chlamydia pneumoniae infection of human coronary atheromas

To date, structures representing developmental stages of Chlamydia pneumoniae, especially persistent forms of this intracellular bacteria, have not been described in human atherosclerotic tissues using specific antibody labeling and transmission electron microscopy.Staining of atherosclerotic tissue from five patients seeking heart transplantation with gold-labeled antibodies specific for up-regulated chlamydial heat shock proteins, GroEL and GroES, and visualisation via transmission electron microscopy revealed intracellular, atypical, round to oval structures of variable diameter. These structures resembled reticulate bodies of Chlamydia, were surrounded by membranes and were located within smooth muscle cells, macrophages or fibroblasts. By using double immunogold electron microscopy technique (GroEL and GroES in combination with chlamydial LPS/MOMP antibodies), we demonstrated these structures were of chlamydial origin.In the current study, we demonstrated the presence of aberrant bodies of C. pneumoniae in vivo in archival coronary atheromatous heart tissues by the immunogold electron microscopy technique.     

Platelet-activating factor inhibits the secretion of platelet-activating factor acetylhydrolase by human decidual macrophages

To clarify the role of platelet-activating factor (PAF) in parturition, the effects of PAF on the secretion of PAF-acetylhydrolase (PAF-AH), a PAF-inactivating enzyme, by decidual macrophage populations were examined. The cells were isolated from human decidual tissue by enzymatic digestion, Ficoll-Paque centrifugation, or flow cytometric sorting. The nonhydrolyzable agonist of PAF, carbamyl-PAF (C-PAF), inhibited the secretion of PAF-AH by either decidual cells or flow cytometrically purified decidual macrophages. A specific PAF receptor antagonist, WEB 2086, blocked the C-PAF-induced inhibition. Lyso-PAF, a metabolite of PAF, had no effect on the enzyme secretion. An intracellular calcium channel blocker, bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid, tetra(acetoxymethyl)-ester, partially blocked the inhibition by C-PAF, whereas extracellular calcium channel blockers, nifedipine and verapamil, were without effect. The inhibitory effect of C-PAF was also partially blocked by protein kinase C (PKC) inhibitors, sphingosine and H-7. A PKC activator, 12-O-tetradecanoylphobol 13-acetate, decreased the secretion of PAF-AH. The decrease was abolished by the addition of sphingosine and H-7. It is suggested that PAF inhibits the PAF-AH secretion by decidual macrophages and that the inhibitory action is mediated by a signal transduction mechanism involving intracellular calcium and PKC.     

Detection and differentiation of Chlamydia trachomatis, Chlamydia psittaci, and Chlamydia pneumoniae by DNA amplification

The polymerase chain reaction was used to detect major outer membrane protein (MOMP) gene sequences from the three species of Chlamydia. Using three primer pairs and one restriction enzyme digestion, three distinct genotypes, corresponding to the three species, Chlamydia trachomatis, Chlamydia pneumoniae, and Chlamydia psittaci, were demonstrated. C. trachomatis was amplified by all three primer pairs and the amplified fragment was digested by EcoRI. C. pneumoniae was amplified by only two of the three primer pairs, and the amplified fragment was digested by EcoRI. C. psittaci was amplified by only two of the pairs and the amplified fragment was EcoRI-resistant. C. trachomatis was detected in direct patient specimens, tissue culture specimens, and fixed specimens, and all serovars of C. trachomatis were detectable. The polymerase chain reaction can detect and differentiate the three species of Chlamydia and may prove a valuable diagnostic tool.     

Chlamydia pneumoniae survival in macrophages is regulated by free Ca2+ dependent reactive nitrogen and oxygen species

OBJECTIVES: Despite an efficient macrophage immune capability, Chlamydia pneumoniae infects host cells and causes chronic diseases. To gain better insights into C. pneumoniae survival mechanisms in macrophages, its growth in regular RAW-264.7 cells (nitric oxide sufficient NO (+)) and RAW-264.7 cells (nitric oxide insufficient NO (-)) were studied. METHODS: Role of Ca(2+), NO and reactive oxygen species (ROS) during C. pneumoniae infection in macrophages were determined. RESULTS: RAW-264.7 NO (-) cells supported significantly Chlamydia growth, showing an upregulation of ROS, superoxide dismutase (SOD) and catalase activities as compared with RAW-264.7 NO (+) cell. Ascorbic acid, inducible nitric oxide synthase inhibitor and glutathione significantly prompted Chlamydia inclusion formation. Cytosolic Ca(2+) had regulatory effect on organism growth, NO generation, SOD and catalase activities in both cell types. CONCLUSIONS: These findings suggest that minimal Ca(2+) signaling in macrophages at early stages of infection, NO and ROS release have modulatory effects onC. pneumoniae survival, onset of persistence and chronicity, processes which are needed for the initiation of diseases in which C. pneumoniae has been implicated as a possible etiologic agent.     

Cultivation of macrophages derived from human malignant effusions

Macrophages, isolated from malignant effusions, were cultured in vitro in media containing GCT/CM as a source of granulocyte-macrophage (GM) colony-stimulating factor (CSF). Cells were periodically examined for macrophage-specific cell surface antigens and functional activity. The number of macrophages increased sixfold during 15 days in culture. Autoradiographic studies confirmed that macrophages were incorporating 3H-Tdr. Macrophage-specific cell surface markers were retained up to five weeks in culture. Cells were also able to express macrophage-specific functions including phagocytosis and secretion of lysozyme. The results indicate that human tumor-derived macrophages may be expanded in short-term culture in the presence of GM CSF.