Carboxypeptidases cathepsins X and B display distinct protein profile in human cells and tissues

Cathepsin X, a recently discovered lysosomal cysteine protease, shares common structural features and activity properties with cysteine protease cathepsin B. Based on its widespread mRNA distribution in primary tumors and tumor cell lines, a redundant function in tumor progression has been proposed. In this study, we have shown that these two related proteases exhibit different profiles with respect to their protein distribution in cells and tissues and to their possible roles in malignancy. Protein level of cathepsin X did not differ significantly between matched pairs of lung tumor and adjacent lung tissue obtained from patients with lung cancer whereas that of cathepsin B was 9.6-fold higher in tumor compared to adjacent lung tissue. Immunohistochemical analysis of lung tumor cathepsin X revealed very faint staining in tumor cells but positive staining in infiltrated histiocytes, alveolar macrophages, bronchial epithelial cells, and alveolar type II cells. Cathepsin X stained positive also in CD68+ cells in germinal centers of secondary follicles in lymph nodes, corresponding to tingible body macrophages. Two cell lines with proven invasive behavior, MCF-10A neoT and MDA-MB 231, showed positive staining for cathepsin B, but negative for cathepsin X. We showed that the invasive potential of MCF-10A neoT cells can be impaired by specific inhibitor of cathepsin B but not by that of cathepsin X. Cathepsin X was found in large amounts in the pro-monocytic U-937 cell line, in monocytes and in dendritic cells, generated from monocytes in vitro. Our results show that cathepsin X is not involved in degradation of extracellular matrix, a proteolytic event leading to tumor cell invasion and metastasis. Its expression, restricted to immune cells suggests a role in phagocytosis and the regulation of immune response.     

Phagocytosis of the Legionnaires' disease bacterium (Legionella pneumophila) occurs by a novel mechanism: engulfment within a pseudopod coil

Phagocytosis of Legionella pneumophila, a bacterial pathogen that multiplies intracellularly in human mononuclear phagocytes and causes Legionnaires' disease, occurs by a novel mechanism. A phagocyte pseudopod coils around the bacterium as the organism is internalized. Human monocytes, alveolar macrophages, and polymorphonuclear leukocytes all phagocytize L. pneumophila by this unusual process, termed "coiling phagocytosis," and these leukocytes phagocytize not only live L. pneumophila in this way, but also formalin-killed, glutaraldehyde-killed, and heat-killed L. pneumophila. In contrast, under the same experimental conditions, monocytes phagocytize Streptococcus pneumoniae, encapsulated and unencapsulated E. coli, Pseudomonas aeruginosa, Pseudomonas alcaligenes, Neisseria gonorrhoeae, and Neisseria meningitidis by conventional phagocytosis. Treatment of L. pneumophila with high-titer anti-L. pneumophila antibody abolishes coiling phagocytosis; such bacteria are internalized by conventional phagocytosis. These experiments raise the possibility that a surface component of L. pneumophila mediates the unusual response by the phagocyte. Such a component, if elaborated in vivo, might be responsible for extrapulmonary manifestations of Legionnaires' disease suspected of being toxin-mediated.     

Metabolism of normal and modified low-density lipoproteins by macrophage cell lines of murine and human origin

Four murine macrophage-like continuous cell lines (P388D1, J774.1, RAW 264.7, and PU5-1.8) and two human cell lines displaying macrophage-monocyte characteristics (HL-60, U-937) have been examined for their ability to degrade both normal and acetylated low-density lipoproteins. All of these cell lines, except PU5-1.8, were demonstrated to have LDL receptors that were induced 2-5-fold by preincubation in lipoprotein-deficient serum. Metabolism of dextran sulfate-LDL complexes by all lines except PU5-1.8 was observed. Three cell lines, P388D1, J774.1 and RAW 264.7, while exhibiting individual differences in their metabolism of acetyl-LDL, all processed acetyl-LDL in a fashion qualitatively analogous to that by murine peritoneal macrophages and human monocytes. Cell lines PU5-1.8, U-937 and HL-60 did not bind or degrade significant quantities of acetyl-LDL. In P388D1 cells, metabolism of acetyl-LDL exhibited time and concentration dependence, was reversibly inhibited by chloroquine, blocked by fucoidan and dextran sulfate, and was calcium independent. Approximately 4 X 10(5) receptors, with an apparent Kd of 3 X 10(-8) M, were present on P388D1 cells. P388D1 cells metabolized 30% as much acetyl-LDL as murine peritoneal macrophages at 37 degrees C and bound 60% as much at 4 degrees C. Chemical measurement demonstrated a 250-fold increase in the cholesteryl ester content of P388D1 cells over 96 h. The accumulation of cholesteryl esters was reversible in the presence of HDL3 and involved continuous hydrolysis and reesterification. These lines represent a convenient resource for examining the metabolism of chemically modified lipoproteins, for isolation of cell mutants, and for isolation of specific lipoprotein receptors.     

The effects of macrophage source on the mechanism of phagocytosis and intracellular survival of Leishmania

Leishmania spp. protozoa are obligate intracellular parasites that replicate in macrophages during mammalian infection. Efficient phagocytosis and survival in macrophages are important determinants of parasite virulence. Macrophage lines differ dramatically in their ability to sustain intracellular Leishmania infantum chagasi (Lic). We report that the U937 monocytic cell line supported the intracellular replication and cell-to-cell spread of Lic during 72?h after parasite addition, whereas primary human monocyte-derived macrophages (MDMs) did not. Electron microscopy and live cell imaging illustrated that Lic promastigotes anchored to MDMs via their anterior ends and were engulfed through symmetrical pseudopods. In contrast, U937 cells bound Lic in diverse orientations, and extended membrane lamellae to reorient and internalize parasites through coiling phagocytosis. Lic associated tightly with the parasitophorous vacuole (PV) membrane in both cell types. PVs fused with LAMP-1-expressing compartments 24?h after phagocytosis by MDMs, whereas U937 cell PVs remained LAMP-1 negative. The expression of one phagocytic receptor (CR3) was higher in MDMs than U937 cells, leading us to speculate that parasite uptake proceeds through dissimilar pathways between these cells. We hypothesize that the mechanism of phagocytosis differs between primary versus immortalized human macrophage cells, with corresponding differences in the subsequent intracellular fate of the parasite.     

Differential regulation of 4E-BP1 and 4E-BP2, two repressors of translation initiation, during human myeloid cell differentiation

Human myeloid differentiation is accompanied by a decrease in cell proliferation. Because the translation rate is an important determinant of cell proliferation, we have investigated translation initiation during human myeloid cell differentiation using the HL-60 promyelocytic leukemia cell line and the U-937 monoblastic cell line. A decrease in the translation rate is observed when the cells are induced to differentiate along the monocytic/macrophage pathway or along the granulocytic pathway. The inhibition in protein synthesis correlates with specific regulation of two repressors of translation initiation, 4E-BP1 and 4E-BP2. Induction of HL-60 and U-937 cell differentiation into monocytes/macrophages by IFN-gamma or PMA results in a dephosphorylation and consequent activation of 4E-BP1. Dephosphorylation of 4E-BP1 was also observed when U-937 cells were induced to differentiate into monocytes/macrophages following treatment with retinoic acid or DMSO. In contrast, treatment of HL-60 cells with retinoic acid or DMSO, which results in a granulocytic differentiation of these cells, decreases 4E-BP1 amount without affecting its phosphorylation and strongly increases 4E-BP2 amount. Taken together, these data provide evidence for differential regulation of the translational machinery during human myeloid differentiation, specific to the monocytic/macrophage pathway or to the granulocytic pathway.     

Non-opsonic phagocytosis of homologous non-toxigenic and toxigenic Corynebacterium diphtheriae strains by human U-937 macrophages

As interactions between bacteria and macrophages dictate the outcome of most infectious diseases, analyses of molecular mechanisms of non-opsonic phagocytosis should lead to new approaches for the prevention of diphtheria and systemic Corynebacterium diphtheriae infections. The present study aimed to evaluate human macrophage–bacteria interactions in the absence of opsonin antibodies and the influence of the tox gene on this process. Homologous C. diphtheriae tox + and tox – strains were evaluated for adhesion, entering and survival within U-937 human macrophages at different incubation periods. Higher numbers of viable bacteria associated with and internalized by macrophages were demonstrated for the tox + strain. However, viable intracellular bacteria were detected at T-24 hr only for the tox – strain. Cytoskeletal inhibitors, cytochalasin E, genistein and colchicine, inhibited intracellular viability of both strains at different levels. Bacterial replication was evidenced at T-24 hr in supernatants of monolayers infected with the tox – strain. Host cell death and nuclear alterations were evidenced by the Trypan blue exclusion assay and DAPI fluorescence microscopy. ELISA of histone-associated DNA fragments allowed detection of apoptosis and necrosis induced by tox + and tox – strains at T-1 hr and T-3 hr. In conclusion, human macrophages in the absence of opsonins may not be promptly effective at killing diphtheria bacilli. The presence of the tox gene influences the susceptibility of C. diphtheriae to human macrophages and the outcome of non-opsonic phagocytosis. C. diphtheriae strains exhibit strategies to survive within macrophages and to exert apoptosis and necrosis in human phagocytic cells, independent of the tox gene.     

Expression of the Opc protein correlates with invasion of epithelial and endothelial cells by Neisseria meningitidis

Whereas capsulate strains of Neisseria meningitidis are dependent on pili for adhesion to human endothelial and epithelial cells, strains which lacked assembled pili and were partially capsule-deficient adhered to and invaded human endothelial and epithelial cells if they expressed the Opc protein. Bacteria expressing low or undetectable levels of Opc protein failed to adhere to or invade eukaryotic cells. In addition, the presence of OpaAC751 protein on the surface of bacteria did not increase bacterial interactions with host cells. Association of Opc-expressing bacteria was inhibited by antibodies against Opc. Invasion was dependent on the host-cell cytoskeletal activity and was inhibited by cytochalasin D. In some cells, infected at the apical surface, bacteria emerging from basal surface were detected by electron microscopy. Opc is found in diverse meningococci and may represent a common virulence factor which facilitates adherence and invasion by these bacteria.     

Fibronectin mediates Opc-dependent internalization of Neisseria meningitidis in human brain microvascular endothelial cells

A central step in the pathogenesis of bacterial meningitis caused by Neisseria meningitidis (the meningococcus) is the interaction of the bacteria with cells of the blood-brain barrier. In the present study, we analysed the invasive potential of two strains representing hypervirulent meningococcal lineages of the ET-5 and ET-37 complex in human brain-derived endothelial cells (HBEMCs). In contrast to previous observations made with epithelial cells and human umbilical vein-derived endothelial cells (HUVECs), significant internalization of encapsulated meningococci by HBMECs was observed. However, this uptake was found only for the ET-5 complex isolate MC 58, and not for an ET-37 complex strain. Furthermore, the uptake of meningococci by HBMECs depended on the presence of human serum, whereas serum of bovine origin did not promote the internalization of meningococci in HBMECs. By mutagenesis experiments, we demonstrate that internalization depended on the expression of the opc gene, which is present in meningococci of the ET-5 complex, but absent in ET-37 complex meningococci. Chromatographic separation of human serum proteins revealed fibronectin as the uptake-promoting serum factor, which binds to HBMECs via alpha 5 beta 1 integrin receptors. These data provide evidence for unique molecular mechanisms of the interaction of meningococci with endothelial cells of the blood-brain barrier and contribute to our understanding of the pathogenesis of meningitis caused by meningococci of different clonal lineages.     

The HL-60 model for the interaction of human macrophages with the Legionnaires' disease bacterium

The facultative intracellular pathogen, Legionella pneumophila, multiplies within and kills human monocytes and alveolar macrophages. We show that L. pneumophila strain Philadelphia-1 infects, multiplies within and kills the promyelocyte HL-60 cell line after its differentiation into macrophage-like cells. The characteristics of the interaction between L. pneumophila and differentiated HL-60 cells closely resemble those between L. pneumophila and human peripheral blood monocytes. With both cell types, C receptors and serum C mediate attachment of L. pneumophila, which are taken up by coiling phagocytosis. The replicative phagosome is lined with ribosomes; intracellular multiplication is iron-dependent; and replicating bacteria ultimately destroy the host cell. As in human monocytes, an avirulent mutant derivative of L. pneumophila Philadelphia-1, 25D, does not replicate in and is not cytopathic for differentiated HL-60 cells. Differentiated HL-60 cells therefore provide a convenient and faithful model for the study of L. pneumophila-mononuclear phagocyte interaction.     

Neisseria meningitidis producing the Opc adhesin binds epithelial cell proteoglycan receptors

Neisseria meningitidis possesses a repertoire of surface adhesins that promote bacterial adherence to and entry into mammalian cells. Here, we have identified heparan sulphate proteoglycans as epithelial cell receptors for the meningococcal Opc invasin. Binding studies with radiolabelled heparin and heparin affinity chromatography demonstrated that Opc is a heparin binding protein. Subsequent binding experiments with purified ³⁵SO₄-labelled epithelial cell proteoglycan receptors and infection assays with epithelial cells that had been treated with heparitinase to remove glycosaminoglycans confirmed that Opc-expressing meningococci exploit host cell-surface proteoglycans to gain access to the epithelial cell interior. Unexpectedly, Opa28-producing meningococci lacking Opc also bound proteoglycans. These bacteria also bound CEA receptors in contrast to the Opc-expressing phenotype, suggesting that Opa28 may possess domains with specificity for different receptors. Opa/Opc-negative meningococci did not bind either proteoglycan or CEA receptors. Using a set of genetically defined mutants with different lipopolysaccharide (LPS) and capsular phenotype, we were able to demonstrate that surface sialic acids interfere with the Opc–proteoglycan receptor interaction. This effect may provide the molecular basis for the reported modulatory effect of capsule and LPS on meningococcal adherence to and entry into various cell types.     

Phenotypic differentiation patterns of the human monocyte/macrophage system

Summary In the present study phenotypic properties of non-stimulated and stimulated blood monocytes and of their normal macrophage derivatives were studied applying enzyme cytochemistry, isoenzyme analysis of acid esterase (EC 3.1.1.6), and immunohistochemical staining using a panel of newly established monoclonal antibodies specific for the monocyte/macrophage lineage. Certain marker profiles could be established for the various normal subpopulations within the monocyte/macrophage system, which were also observable in epithelioid cells and U-937 cell line considered as reactive and neoplastic differentiation variants of monocytes, respectively. Alveolar macrophages, in contrast to the other analysed monocyte/macrophage populations, showed a highly activated phenotype comparable to lymphokine stimulated blood monocytes and epithelioid cells. The results underline the concept that the adaptation of monocytes/macrophages to their particular microenvironment is of decisive importance for their definitive differentiation.     

Serglycin and secretion in human monocytes

The human monocytic cell line U-937 has been widely used as a model system for human monocytes. The subclone U-937-B has been adapted to serum-free conditions. This particular U-937 clone and its parent clone U-937-1 were used to investigate the role of the proteoglycan serglycin in human monocytes. For this purpose cells were treated with hexyl-β-D-thioxyloside to abrogate proteoglycan expression. U-937-B cells expressed and secreted exclusively chondroitin sulphate proteoglycans, and after treatment with this xyloside they only expressed and released free chondroitin sulphate chains. Western blotting showed that serglycin core protein was present in conditioned medium of control cells, but absent in medium from xyloside-treated cells. Also, serglycin core protein could be detected in the cell fractions of control cells, but not in the cell fractions from xyloside-treated cells. Furthermore, less proteoglycan-associated proteins could be detected in medium from cells incubated with xyloside, suggesting that the absence of secreted sergycin affects the secretion of such proteins. Cells incubated in the presence of xyloside were analyzed by transmission electron microscopy and shown to contain numerous large empty vesicles. The lack of serglycin, the dominant proteoglycan in U-937 monocyte-like cells, consequently, leads to effects on vesicle formation and secretion of some low molecular weight proteins, suggesting that this particular proteoglycan is of importance for secretory processes in human monocytes.     

OmpD but not OmpC is involved in adherence of Salmonella enterica serovar typhimurium to human cells

Conflicting reports exist regarding the role of porins OmpC and OmpD in infections due to Salmonella enterica serovar Typhimurium. This study investigated the role of these porins in bacterial adherence to human macrophages and intestinal epithelial cells. ompC and ompD mutant strains were created by transposon mutagenesis using P22-mediated transduction of Tn10 and Tn5 insertions, respectively, into wild-type strain 14028. Fluorescein-labeled wild-type and mutant bacteria were incubated with host cells at various bacteria to cell ratios for 1 h at 37 degrees C and analyzed by flow cytometry. The mean fluorescence intensity of cells with associated wild-type and mutant bacteria was used to estimate the number of bacteria bound per host cell. Adherence was also measured by fluorescence microscopy. Neither assay showed a significant difference in binding of the ompC mutant and wild-type strains to the human cells. In contrast, the ompD mutant exhibited lowered binding to both cell types. Our findings suggest that OmpD but not OmpC is involved in the recognition of Salmonella serovar Typhimurium by human macrophages and intestinal epithelial cells.     

Interleukin-1 and tumour necrosis factor production by human monocytoid cells: study on a single cell level.

Human IL-1 beta and TNF alpha production by normal and transformed monocytoid cells was studied using biological assays, cytokine specific ELISA and by immunocytochemical methods on a single cell level. Quiescent human blood monocytes and cultured in vitro transformed human monocytoid cell lines U-937, THP-1 and HL-60 did not contain IL-1 beta and TNF alpha in their cytoplasm. IL-1 beta synthesis and secretion was induced by LPS stimulation in nearly 90% monocytes, 15-20% U-937, 3-5% THP-1 and in no HL-60 cells. Normal human blood monocytes had a more rapid kinetics of IL-1 beta synthesis. IL-1 beta positive cells stained with antibodies to human IL-1 beta appeared at 1-2 hours after LPS application, while in monocytic cell lines only after 4-6 hours. Using immunoperoxidase staining of U-937 cells pulse labelled with 3H-thymidine, it was shown that proliferating cells did not synthetize IL-1 beta. Instead of IL-1 beta, TNF alpha could be induced by LPS in U-937 cells only after preliminary differentiation with PMA. Recombinant IL-1 beta induced a very low level of TNF alpha production in PMA-treated cells. Similarly recombinant TNF alpha alone induced IL-1 beta synthesis only in a few U-937 cells.     

Interleukin-8 secretion of human epithelial and monocytic cell lines induced by middle ear pathogens

Otitis media with effusion (OME) is one of the most common diseases in children. Alloiococcus otitidis, a new gram-positive bacterial species, was isolated from the middle ear fluid of children with OME; however, the pathogenic role of this bacteria is yet unknown. In this study, the ability of cultured epithelial cell lines (Hep-2 and Hela) and monocytic cell lines (THP-1 and U 937) to secrete chemokine interleukin-8 (IL-8) in response to the A. otitidis organism and three bacterial organisms mainly detected from middle ear fluid in OME, and bacterial cell components was investigated. When stimulated with four viable bacterial cells, epithelial cells and monocytes secreted IL-8 in a time-dependent manner. The monocytes produced significantly higher levels of IL-8 than the epithelial cells. Compared with that by viable bacterial cells, IL-8 secretion by stimulated epithelial cells and monocytes was reduced when the bacteria were heated and treated with glutaraldehyde. With bacterial stimulations, cell treatment of interferon-gamma caused monocytes to increase the induction of IL-8 production, however, the induction of monocyte differentiation caused monocytes to reduce the induction of IL-8 production. Furthermore, epithelial cells and monocytes stimulated by four viable bacterial organisms physically separated from cultured cells reduced the induction of IL-8 compared with directly stimulated cells, and monocytes stimulated with soluble extracts prepared from A. otitidis organisms produced IL-8 in a dose-dependent manner. These results suggest that part of the IL-8 stimulation of the A. otitidis organism may exist in a diffusable factor released by the bacteria or soluble components of the bacteria itself.     

MLC-conditioned media stimulate an increase in Fc receptors on human macrophages

Macrophage Fc receptors (FcR) are essential for antibody-dependent cellular cytotoxicity and for optimal phagocytosis of opsonized particulate antigens. Culture in the presence of conditioned medium from mixed leukocyte cultures (MLC-CM) resulted in a dose- and time-dependent increase (up to 10-fold) in FcR-dependent binding of 125I-labeled IgG1 to promyelocytic HL-60 cells, macrophage-like U-937 cells, and normal cultured human monocytes. FcR increase in HL-60 cells was blocked by cycloheximide (100 microM) and was accompanied by a slight decrease in binding affinity. Since cell volume did not change, the increase in FcR probably represents an increase in the surface density of FcR sites. MLC-CM prepared with or without serum were equally effective in augmenting FcR sites, whereas only serum-containing MLC-CM caused morphologic change of U-937 and HL-60 cells.     

Effects of recombinant human colony stimulating factors (CSF) (granulocyte-macrophage CSF, granulocyte CSF, and CSF-1) on human monocyte/macrophage differentiation

Purified recombinant human granulocyte-macrophage (rhuGM)-CSF, rhuG-CSF, and rhuCSF-1 were evaluated for their capacity to influence the differentiation of U-937 cells and normal human monocytes. The human U-937 cell line represents an early stage of monocytic differentiation. It was found that rhuGM-CSF and rhuG-CSF, but not rhuCSF-1, induced phenotypic changes consistent with monocyte/macrophage differentiation in U-937 cells. After 3 days of culture in the presence of either rhuGM-CSF or rhuG-CSF, a small but significant proportion of U-937 cells were able to reduce nitroblue tetrazolium. Nitroblue tetrazolium reduction, however, was maximally induced when rhuGM-CSF and rhuG-CSF were added in combination. These changes were accompanied by increased alpha-naphthyl acetate esterase activity, acquisition of macrophage morphology, Mo-1 Ag expression, and decreased cell proliferation. rhuGM-CSF alone also induced expression of the c-fms proto-oncogene (CSF-1 receptor) in U-937 cells and this expression was enhanced by the combination of rhuGM-CSF and rhuG-CSF. In cultured normal human peripheral blood monocytes, representing a late stage of maturation, rhuGM-CSF and rhuCSF-1 differentially increased Mo-1 and My-4 Ag expression, respectively, whereas rhuG-CSF was without effect. Our results suggest that the interaction of GM-CSF, G-CSF, and CSF-1 may play a fundamental role in the early and late stages of the human monocyte/macrophage differentiation process.