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PubMed1979

Neutral protease secretion by human monocytes. Effect of surface-bound immune complexes.

The effect of surface-bound immune complexes on the secretion of neutral proteases by human peripheral monocytes was examined. Monocytes cultured on 125I-fibrin secreted plasminogen activator in a continuous fashion. Monocytes incubated on 125I-fibrin with surface-bound immune complexes displayed a burst of plasminogen-independent fibrinolytic activity, whereas no release of plasminogen activator was observed through 21 h. The plasminogen-independent fibrinolytic enzymes were derived from monocytes and not from lymphocytes or contaminating polymorphonuclear neutrophils. The effects of various protease inhibitors on the secretion of plasminogen-dependent and independent enzymes were determined. Chymostatin selectively inhibited the monocyte-derived plasminogen activators. Similar effects of chymostatin were observed on human urokinase in the absence of cells. The predominant protease producing plasminogen-independent fibrinolysis exhibited responses to inhibitors characteristic of leukocyte elastase. When monocytes were cultured on 125I-fibrin with adherent immune complexes approximately equal to 40% of the solubilized radioactivity represented deiodination and not proteolysis. It was concluded that culture of human monocytes on surface-bound immune complexes stimulates the secretion of plasminogen-independent fibrinolytic proteases, primarily elastase, and of deiodinating enzymes. Under these conditions, plasminogen activator secretion is inhibited. Neutral proteases secreted from newly recruited monocytes may contribute to tissue injury in human diseases characterized by the presence of adherent immune complexes.

Antigen-Antibody Complex↗
PubMed1979

Human monocyte-mediated cytotoxicity to K-562 cells: activation by lymphokines.

Human monocytes purified by adherence and prolonged in vitro monolayer culture were activated by supernatants of autologous lymphocytes stimulated with live bacillus Calmette-Guérin or killed Corynebacterium parvum. Activated monocytes expressed increased ability to lyse K-562 cells prelabelled with methyl-3H-thymidine in a 48 h assay. Activation could be detected at a 1:64 dilution of lymphokine supernatants. Target cell killing by activated monocytes was strongly influenced by the density of the monocyte monolayer. Monocytes at an intermediate stage of in vitro differentiation seemed to be most response to lymphokine activation. Morphological studies indicated that a considerable number of K-562 cells attach firmly to monocytes. The monocyte-mediated lysis of K-562 seems to be extracellular, as phagocytosis was not observed. Freshly isolated adherent blood mononuclear cells (greater than 90% monocytes) were strongly cytostatic to K-562 cells, as determined by inhibition of methyl-3H-thymidine uptake. Considerable cytolytic activity was also found with freshly isolated adherent cells, strong enhancement being produced by performing the assay in the presence of newborn calf serum, as opposed to human AB serum.

Cell Differentiation↗
PubMed1977

Computer-assisted monocyte esterase assay by flow-cytophotometry.

A Wang model 2200 computer has been interfaced with the Bio/Physics Systems, Inc. model 6300 Cytograf and model 2100 Distribution Analyzer. Using a custom designed software program, in conjunction with an azo-dye technic for staining monocytes for nonspecific esterase activity, it has been possible to obtain rapid and reliable data concerning relative values for intracellular monocyte esterase activity. The method is based on measuring the axial light-loss voltage signal for each of one thousand stained monocytes. Individual stained monocytes were assigned to one of four groups (A, B, C, D), dependent upon the magnitude of the signal and were given different rating values (1, 2, 3, 4) according to their group designation. A "score" was derived for each blood sample by multiplying the percentage of cells (monocytes) in each group category by the appropriate factor and summing these values. The technic permits rapid objective assessment of intracellular nonspecific esterase activity in monocytes suspended in a mixed cell population. Both Gaussian and bi-modal patterns for monocyte esterase were observed. The latter suggests a dual monocyte population.

Computers↗
PubMed1979

Cytostatic effect on tumour cells induced in human monocytes by mediators from BCG-stimulated lymphocytes and MLC.

Human monocytes activated by mediators (lymphokines) from BCG-stimulated, sensitized lymphocytes (from BCG-vaccinated donors) were cytostatic to a human cell line. Mediators from allogeneic lymphocytes activated the cytostatic ability of monocytes to the same degree as mediators from autologous lymphocytes. Mediators from BCG-stimulated lymphocytes from tuberculin-negative donors not vaccinated with BCG, activated the monocytes only to a small extent. Culture of lymphocytes in a membrane chamber (MC) proximate to monocytes, or incubation of monocytes with filtered supernatants of lymphocyte cultures, were equally effective procedures for inducing cytostatic ability in monocytes. Supernatants of sensitized lymphocytes cultured on BCG for four hours did not activate the monocytes, while supernatants collected after 24 hours activated the cytostatic ability to the same extent as 72-hour supernatants. Supernatants of mixed lymphocyte cultures (MLC) collected after 24 hours did not activate the monocyte cytostatic ability at all. Forty-eight and 72-hour supernatants of MLC showed a small but increasing activity. There was no significant difference between BCG-stimulated lymphocytes and MLC in their maximum DNA synthesis or in the kinetics of their DNA synthesis. Thus, the DNA synthesis and secretion of lymphocyte mediators may be independent phenomena resulting from the same stimulus.

BCG Vaccine↗
PubMed1979

In vitro influence of endotoxin on human mononuclear phagocyte structure and function. 2. Enhancement of the expression of cytoststic and cytolytic activity of normal and lymphokine-activated monocytes.

The presence of non-toxic concentrations of E. coli endotoxin (LPS) during the in vitro interaction of normal human monocytes and a human tumour cell line (NHIK 3025) enhanced monocyte-mediated target cell cytostasis and cytolysis. Monocyte responsiveness to LPS was greatest at an intermediate stage of in vitro differentiation. The expression of cytostatic and cytolytic activity by human monocytes activated with mediators from Corynebacterium parvum-stimulated human lymphocytes was also enhanced by LPS. Lymphokine activation did not induce additional LPS responsiveness in the monocytes. Monocytes activated with lymphokines and subsequently deactivated by in vitro culture did not show any increase in LPS responsiveness. A soluble cytostatic factor, which is probably not cold thymidine, was released from monocytes exposed first to lymphokines and then to LPS. While LPS is ineffective as an induction signal of monocyte cytotoxicity to tumour cells in this system, it enhances the expression of cytotoxicity induced by prolonged in vitro culture or lymphokine activation.

Adult↗
PubMed1979

Separation of and cholesterol synthesis by human lymphocytes and monocytes.

We have devised techniques for the isolation of human monocytes which do not require the adherence of the cells to a surface. In 15 consecutive experiments using density-gradient and counterflow centrifugations, a population of mononuclear cells that was 75 +/- 11% monocytes was obtained within 2 hours of venipuncture. These cells had never been pelleted and represented approximately three-fourths of the monocytes that had been present in the whole blood. In another 22 consecutive experiments using sedimentation in gelatin followed by counterflow and density-gradient centrifugations, a population of lymphocytes that was 99.5 +/- 0.5% pure and a population of monocytes that was 94 +/- 3% pure were obtained within 3 hours of venipuncture. When these freshly isolated cells were incubated in the lipoprotein-deficient fraction of serum (d > 1.21 g/ml) or in solvent-extracted serum, the monocytes incorporated 10-20 times more [2-(14)C]acetate into sterols than did the lymphocytes. Monocytes were seen to constitute between 6 and 46% of the mononuclear cells isolated from normal individuals by the usual density-gradient centrifugation of whole blood on Ficoll-Hypaque. We conclude that future studies of cholesterol metabolism utilizing human mononuclear cells must take into account this large variation in the percentage of monocytes and their disproportionately greater activity during short-term incubations in media that induce sterol synthesis.-Fogelman, A. M., J. Seager, M. Hokom, and P. A. Edwards. Separation of and cholesterol synthesis by human lymphocytes and monocytes.

Adult↗
PubMedDate not supplied

Cytochemical heterogeneity of human circulating monocytes.

Normal human monocytes, based on appearance after Romanowsky-type staining, have been considered to be a homogenous class of mononuclear leukocytes. Cytochemical studies, however, indicate that these cells are markedly heterogenous with respect to certain hydrolytic and oxidative lysosomal enzymes and to a lesser extent show variability in cytoplasmic carbohydrate and ribonucleic acid content. Individual monocytes were found to vary most widely in non-specific esterase activity and least in acid phosphatase activity. Variation in monocyte peroxidase activity was intermediate between these extremes. Using a cytochemical "scoring" technic, younger adults were found to have higher mean monocyte non-specific esterase scores than older adults. No significant age or sex differences were found for peroxidase or acid phosphatase activity. Furthermore, the overall activities of these monocyte enzymes in healthy adults, as estimated by intensity of staining, were unrelated to one another. Using consecutive double staining technics, individual monocytes were also found to vary in their content of these biochemical consituents. A statistically inverse relationship was found in individual monocytes between their nonspecific esterase and acid phosphatase activities (r = -0.68). Analysis of other combinations studied showed only random distribution with no significant correlations. These findings emphasize the biochemical individuality of circulating monocytes and the variations noted probably reflect differences in number and composition of lysosomes.

Acid Phosphatase↗
PubMed1977

Stimulation of monocyte procoagulant activity by adherence to different surfaces.

During in vitro incubation for several hours human circulating blood leucocytes developed procoagulant activity (PCA) which was identified as tissue thromboplastin-like activity and which remained specifically associated with monocytes. Adherence of monocytes to glass stimulated these cells to generate PCA. When cuprophane was used, both the degree of adherence of monocytes and PCA generation by these cells was significantly less. In addition, when the monocytes were incubated upon a monolayer of cultured endothelial cells only minor PCA generation was detectable. After incubation on glass the non-adherent monocytes had negligible PCA in comparison to the adherent monocytes. Protein synthesis inhibitors (cycloheximide and actinomycin D) inhibited PCA generation but did not affect monocyte adherence. These results demonstrate that adherence of monocytes is a stimulus for the generation of PCA.

Blood Coagulation↗
PubMed1978

Human monocyte cytotoxicity to tumor cells. I. Antibody-dependent cytotoxicity.

Recent investigations examining mononuclear cell antibody-dependent cell-mediated cytotoxicity against tumor cell lines suggest that K lymphocytes and not monocytes are active in this cytotoxic reaction. We have found, however, that in an allogeneic assay system, human monocyte monolayers as well as lymphocytes mediate substantial lysis of 51Cr-labeled antibody-coated CEM lymphoblast tumor cells. This cytotoxicity is temperature-dependent and rapid, with most 51Cr release occurring in the first 4 hr of co-incubation. Interaction between target cell-bound antibody and the monocyte Fc receptor is necessary as demonstrated by the marked fall in antibody-dependent cell-mediated cytotoxicity (ADCC) produced by staphylococcal protein A, high concentrations of nonspecific immunoglobulin, and dilution of the target cell antiserum. Morphologic and functional characteristics of the monocyte-monolayer preparations establish their relative purity (greater than 95%) and indicate that monocytes and not contaminating lymphocytes are responsible for tumor cell lysis. Furthermore, preincubation of monocyte and lymphocyte preparations with latex particles or low concentrations of immunoglobulin distinguished monocyte from lymphocyte ADCC. Thus, normal human monocytes have the capacity to carry out antibody-dependent cytotoxicity against nucleated malignant target cells.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1977

The monocyte as a model for the study of insulin receptors in man.

We have characterized the cellular composition of preparations isolated from peripheral blood by Ficoll-Isopaque gradient centrifugation. 125I-insulin binding to every cell type was measured. A highly significantly positive correlation between specific cell binding fraction and the monocyte concentration of the heterogeneous cell suspension was demonstrated. Depletion of monocytes reduced the insulin binding approximately 80%, which confirms previous findings by other investigators. The granulocytes possessed the second highest binding ability, but only one fourteenth of that of monocytes. Compared to the lymphocyte the monocyte had about 25 times greater insulin binding. Also thrombocytes bound insulin and contamination with these meant that their contribution to the total specific cell binding was not negligible. A reduction in these contaminants is essential. We found that insulin binding to erythrocytes was insignificant. A method of calculating the specific insulin binding to monocytes alone is introduced. The monocyte-insulin-receptor possesses specificity. Only an insignificant degradation of receptor bound insulin could be shown. Evidence of negative cooperativity between receptors was found. Consequently monocytes are considered a useful model for insulin receptor studies in man.

Binding, Competitive↗
PubMed1977

Two-step separation of human peripheral blood monocytes on discontinuous density gradients of colloidal silica-polyvinylpyrrolidinone.

Normal human peripheral blood monocytes were purified by a two-step separation. The first step, the standard Ficoll--Hypaque (F--H) buoyant density centrifugation, yielded mainly mononuclear cells, of which 24 +/- 9% were monocytes. Isopycnic centrifugation on discontinuous gradients of colloidal silica polyvinylpyrrolidinone (CS-PVP) further separated these mononuclear cells. The density interface between 1.070 and 1.060 g/ml yielded 82 +/- 7% monocytes, 5 +/- 4% granulocytes and 13 +/- 8% lymphocytes. Sixty-six percent of the monocytes obtained after F--H separation were recovered in this layer. The monocytes were intact and viable and retained their ability to phagocytose and kill Candida pseudotropicalis and to spread on glass coverslips. Motility (both random and towards a chemoattractant) was retained but was quantitatively less than after F--H separation alone. The relative purity of the monocyte population allowed assessment of major histocompatibility surface antigens by serotyping. This confirmed the presence of HLA and Ia-like antigens on monocytes.

B-Lymphocytes↗
PubMed1978

Normal human monocytes inhibit tumor cell growth in vitro.

Normal human monocytes were evaluated in an in vitro assay of growth inhibition of tumor cells. Monocytes were isolated from the blood of 6 normal subjects by Ficoll-Hypaque separation and adherence to plastic microtest wells. Cervical carcinoma cells (HeLa) were added to the microwells to result in a ratio of 50 monocytes to one HeLa cell. Cultures were then incubated for 6 to 46 hr. Growth inhibition was evaluated by measuring the uptake of 3H-thymidine over a 4-hr pulse period after 2, 18, or 42 hr of monocyte-HeLa interaction. Inhibition of HeLa growth by monocytes was 23.8% +/- 8.6% over 6 hr, 22.0% +/- 8.9% over 22 hr. and 68.3% +/- 7.5% over 46 hr. Growth inhibition of HeLa cells was confirmed by direct enumeration of HeLa cells at the end of coincubation. Attachment of monocytes to the HeLa cells was confirmed by light and scanning electron micrographs. Granulocytes, lymphocytes, and other cell lines did not comparably inhibit HeLa growth and media replenishment did not ablate the effect. These data demonstrate that normal human monocytes can inhibit the growth of a malignant cell line in vitro in the absence of overt activation procedures.

Cell Division↗
PubMed1978

Requirement of S-adenosyl-L-methionine-mediated methylation for human monocyte chemotaxis.

The chemotactic response of motile bacteria requires the methylation of specific proteins by S-adenosyl-L-methionine. To determine whether methylation is required for the chemotaxis of human leukocytes, we studied the effects of inhibition of S-adenosyl-L-methionine-mediated methylation on monocyte chemotactic responsiveness. Methylation was inhibited in monocytes by treating the cells with substances that produced elevations in intracellular S-adenosyl-L-homocysteine, a competitive inhibitor of S-adenosyl-L-methionine methylation. Treatment of isolated monocytes with the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine, plus exogenous adenosine and L-homocysteine thiolactone increased intracellular S-adenosyl-L-homocysteine levels by as much as 1500-fold. Concomitant with increases in S-adenosyl-L-homocysteine were a decrease in monocyte protein carboxy-O-methylation as well as a marked inhibition of monocyte chemotactic responsiveness. Conditions that almost completely inhibited methylation and chemotaxis did not depress monocyte phagocytosis, indicating that this latter function either is independent of S-adenosyl-L-methionine-mediated methylation or is extremely resistant to inhibition of such reactions by S-adenosyl-L-homocysteine. These studies indicate that S-adenosyl-L-methionine-mediated methylation is required for the chemotaxis of eukaryotic cells and that the chemotactic and phagocytic functions of human monocytes have different requirements for methylation.

Adenosine Deaminase Inhibitors↗
PubMed1979

Defective leukotaxis in monocytes from patients with pulmonary tuberculosis.

Because the accumulation of macrophages and their precursors, peripheral blood monocytes, in foci of infection is an important feature of the host reponse to mycobacterial challenge, the leukotactic responsiveness of monocytes from patients with active tuberculosis was evaluated. With a double-filter, in vitro technique, defective leukotaxis was demonstrated in monocytes from 19 of 20 untreated patients, whereas normal leukotactic responses were found in monocytes from 11 of 15 patients with chronic, nontuberculous pulmonary inflammatory diseases. This defect may be related to increased activity of a naturally occurring, heat-stable plasma substance with a molecular mass of approximately 2.3 x 10(5) daltons that inhibited leukotactic responsiveness. Monocyte leukotaxis improved and the leukotactic inhibitory activity of plasma disappeared in most patients while they were on therapy; these phenomena were unrelated to bacteriologic conversion or resolution of symptoms. In vitro studies with isoniazid, ethambutol, and rifampin excluded a direct effect of these drugs or their metabolites on monocytes or on the leukotactic inhibitor in plasma. Thus, defective leukotaxis of monocytes in patients with pulmonary tuberculosis may be an epiphenomenon of the local tissue reaction.

Adolescent↗
PubMed1976

Effects of staphylococcal products on locomotion and chemotaxis of human blood neutrophils and monocytes.

The effects of staphylococcal products as chemo-attractants for human blood neutrophils and monocytes and as inhibitors of locomotion of these cells were studied with bacterial cells, culture filtrates and isoelectrically focused fractions from culture filtrates of nine strains of Staphylococcus aureus. Little direct chemotactic activity of staphylococcal products for neutrophils was observed, although a chloroform-soluble extract of the whole organisms contained such activity. The major chemotactic effect of staphylococci for neutrophils was indirect, i.e., generated when the organisms or their products were incubated with plasma, perhaps due to activation of complement. In contrast, direct chemotactic activity for monocytes was found in a large number of staphylococcal fractions. Staphylococci also produced inhibitors of locomotion of both neutrophils and monocytes. Isoelectric focusing showed more fractions inhibitory for neutrophils than for monocytes. Some of the inhibitors could be identified. Staphylococcal alpha-toxin inhibited migration of both neutrophils and monocytes. Sphingomyelinase C (beta toxin) inhibited migration of monocytes but not of neutrophils. Leucocidin-rich strains were strongly active as inhibitors of neutrophil locomotion but less so as inhibitors of monocyte locomotion.

Cell Migration Inhibition↗
PubMed1978

Ultrastructural identification of acid complex carbohydrate in cytoplasmic granules of normal and leukaemic human monocytes.

Complex carbohydrate in granules of monocytes was compared with that in granules of neutrophils by ultrastructural cytochemical methods. The acid mucosubstance in granules of both cell types stained with dialysed iron after brief fixation with dilute glutaraldehyde, but that in monocyte granules differed in failing to stain after stronger fixation. Approximately 10% granules in normal blood monocytes stained with this method, whereas more than 90% of granules in leukaemic monocytes from two of seven patients with acute myelomonocytic leukaemia stained intensely. This difference presumably results from unmasking of acid groups in immature granules or increased synthesis of granule mucosubstance in some leukaemic monocytes. Granules of monocytes differed further from those of neutrophils in failing after either type of fixation to stain with a high iron diamine technique for for demonstration of sulphated mucosubstance. The absence of high iron diamine staining could reflect a lack of sulphate esters in monocyte granule mucosubstance, masking of the sulphate groups by other components, or extraction of the sulphated mucosubstance during specimen processing.

Cytoplasmic Granules↗
PubMed1975

Evaluation ofprimary blood monocyte and bone marrow cell culture for the isolation of Rickettsia rickettsii.

Rickettsia rickettsii was isolated from experimentally infected guinea pigs by culture of blood monocytes and bone marrow cells, and from experimentally infected rhesus monkeys by blood monocyte culture. Rickettsiae were identified in monocyte-macrophage monolayers stained by Giménez or flourescent antibody techniques. A total of 78 culture attempts were made from 20 guinea pigs and 16 monkeys. The success of isolation of R. rickettsii in culture was positively correlated with the numbers of rickettsiae present in the blood and bone marrow. in cultures derived from infected guinea pigs, rickettsiae were usually observed after 5 to 7 days of culture, and in monkeys monocyte cultures they were usually observed within 3 to 5 days. Positive cultures were derived from guinea pigs and monkeys as early as the first day of fever and 1 to 3 days before the appearance of other clinical signs. Monocyte cultures became negative with the resolution of rickettsemia and concomitantly with the appearance of serum antibody. Monocyte culture isolation of R. rickettsii may be as sensitive for the detection of rickettsiae in blood and marrow as the intraperitoneal inoculation of guinea pigs or the plaque assay technique. Because of the simplicity of the method and because rickettsiae were often identified within 3 to 5 days after initiation, the monocyte culture technique may be useful in the early diagnois of human rickettsial disease.

Animals↗
PubMed1979

Enhancement of generation of monocyte tissue thromboplastin by bacterial phagocytosis: possible pathway for fibrin formation on infected vegetations in bacterial endocarditis.

The deposition of fibrin on infected vegetations and the presence of mononuclear phagocytes that have phagocytized bacteria are remarkabe features in experimental bacterial endocarditis. In a study in vitro, we show that phagocytosis of bacteria by human monocytes enhances thromboplastin generation by these cells. Maximal enhancement of the generation of thromboplastin by monocytes was about six times compared with that in the control experiment without bacteria, and it was obtained by preincubation of the monocytes with 5 to 10 bacteria per monocyte. No quantitative difference was observed between Staphylococcus epidermidis and Streptococcus sanguis as to the enhancement of the monocyte thromboplastin generation. An enhancement of the procoagulant activity generation was also observed after addition of bacteria to human or rabbit whole blood. Probably, this generation was also due to synthesis of thromboplastin by monocytes. It is conceivable that fibrin deposition on infected vegetations during bacterial endocarditis is mediated by thromboplastin synthesis by monocytes.

Animals↗