Monocyte Suspensions Research Articles

In vitro release of lysozyme from monocytes and granulocytes.

When exposed to zymosan or latex particles or heat-inactivated staphylococci, freshly prepared human blood monocytes and granulocytes rapidly released a large fraction of their lysozyme content. Within 24 hours the total lysozyme activity in the monocyte suspensions tripled, while it doubled in the granulocyte suspensions, indicating synthesis of the enzyme following release. The monocytes in particular seemed to release and synthesize lysozyme without any other stimulus than contact with lymphocytes and the tube walls. Potassium caseinate in solution did not influence the lysozyme release. Myeloperoxidase and beta-glucuronidase, which in the granulocytes are kept in lysosomal fractions separate from most of the lysozyme, were neither released nor synthesized to a significant degree. Moreover, the minute amount of lactate dehydrogenase released indicated that the lysozyme release was not the result of cell lysis. Accordingly, the monocytes, which are not already stimulated by adherence to nonphagocytosable surfaces, are capable of selective enzyme release similar to that of the granulocytes.

A simple method of obtaining monocytes in suspension

Relatively pure monocyte suspensions may be obtained by density flotation after simple conditioning of leukocytes. This consists of incubating blood or buffy coat cells in plasma containing NaCl at a higher than physiological concentration. As a result the density of granulocytes and lymphocytes increases greatly, but the density of monocytes changes very little. It is possible with one centrifugation on a slightly modified density gradient to obtain 90% pure monocyte suspensions with 55% yield. More than 95% purity but lower yield is obtained simply by increasing the NaCl concentration. The membrane characteristics of monocytes and their capacity to function normally remain unaltered by this treatment and cells so obtained develop in culture into macrophages normally. This method is much cheaper and simpler than others described, and is applicable equally to either small or large numbers of leukocytes.

Improved Monocyte Cytotoxicity Test

A micro-modification of the two-colour monocyte cytotoxicity test (MCTT) is described. It employs exclusively adherent monocytes for staining, sensitizing and. labeling instead of monocyte suspensions. Thereby unspecific cytotoxicity is decreased while sensitivity and specificity of the test are improved. As a micro-technique, this modification of the MCTT is useful for antibody screening as well as antigen definition.

Receptor binding of lactoferrin by human monocytes.

The binding of iron-saturated 125I-lactoferrin to human monocytes was studied at pH 7.4 and 37 degrees C. Monocytes in suspension bound 125I-lactoferrin by a reversible, saturable and specific binding indicating the presence of a receptor. The dissociation constant (KD) of the binding was estimated at about 4.5 x 10(-9) M and the number of receptors was about 1.6 x 10(6) per monocyte. The affinity of native lactoferrin (20% iron saturated) was only slightly below that of iron-saturated lactoferrin (KD about 7.9 x 10(-9) M). Human transferrin, horse cytochrome c and human immunoglobulin G were without inhibitory effect on the binding of 125I-lactoferrin. The majority of cell-bound 125I-lactoferrin was dissociable. The dissociation rate was not affected by addition of unlabelled lactoferrin to the dissociation medium. The binding of 125I-lactoferrin to adherent mononuclear blood cells showed an about 100-fold lower affinity (KD about 2.5 x 10(-7) M) than to cells in suspension, but the specificity of the binding was the same. These results are compatible with the idea that lactoferrin exerts a biological effect mediated by an interaction with cells of the monocyte/macrophage lineage.

Isolation of human blood monocytes with Nycodenz, a new non-ionic iodinated gradient medium.

Monocytes were separated from human blood with Nycodenz, an iodinated gradient medium. Monocytes have a lower average density than lymphocytes, but because of overlapping efficient separation cannot be achieved on the basis of density differences alone. Thus the isolation procedure was based on the assumption that the low-density fraction of lymphocytes increases its density more than monocytes by expelling water when exposed to an increased osmolarity. Thereby they might pass through a density barrier present initially, whereas the monocytes remain at the top of the gradient layer. Separation fluids with densities ranging from 1.061 to 1.096 g/ml were prepared by mixing Nycodenz with NaCl solutions of various concentrations. EDTA-blood (3 ml) or a leucocyte suspension (2-6 ml) obtained by dextran sedimentation was loaded on 3 ml of separation fluid and centrifuged for 15 min at 1900 rpm. Then the cells in the interface region were collected. At each density level it was possible to obtain an almost pure monocyte suspension (95-98%) by increasing the osmolarity. However, the higher the purity, the lower the monocyte yield. Apparently, the viability of monocytes was not affected, even when subjected to an osmolarity of 600 mosmol. For routine use, it appears that separation fluids with densities from 1.061 to 1.078 g/ml and corresponding osmolarity in the 300 to 410 mosmol range are suitable.

Increased antibody-dependent cytotoxicity mediated by purified monocytes in Hodgkin's disease

Monocyte antibody-dependent cytotoxicity was studied in 19 patients with Hodgkin's disease and 14 normal controls. This function was investigated after isolation of the monocytes by means of a modified elutriation technique. Direct sizing and counting of the cells present in the effluent enabled individual adjustment during each separation procedure. The absolute monocyte count in the peripheral blood of patients with Hodgkin's disease was higher ( P < 0.002) than in normal controls. Nearly 90% pure monocyte suspensions, representing 82% of all elutriated monocytes, were obtained. The elutriation characteristics of the monocytes in both groups were essentially the same, irrespective of marked interindividual differences. Kill of antibody-coated chicken red blood cells was measured by DNA flow cytometry. In comparison to normal controls, a significantly increased ( P < 0.0004), stage-independent, monocyte antibody-dependent cytotoxicity was found in patients with Hodgkin's disease. The percentage of kill in symptomatic patients tended to be higher than in the asymptomatic group; no correlation was found with the absolute number of circulating monocytes.

Human factor VII associated with endotoxin stimulated monocytes in whole blood

Evidence is provided for a calcium dependent binding of factor VII to stimulated monocytes in whole blood. The binding of factor VII to the monocytes in this study was directly related to the exposure of tissue thromboplastin on the surface of the endotoxin stimulated monocytes. In such monocytes synthesis of tissue thromboplastin is well known to be enhanced. Antibodies against the apoprotein of tissue thromboplastin prevented the appearance of factor VII activity in monocyte suspensions isolated from blood incubated with endotoxin. This could be explained by the failure of factor α-VIIa generation in blood containing thromboplastin antibodies. Factor α-VIIa is probably the form of factor VII being bound to the monocytes.

Esterase staining of monocytes in suspensions of Ficoll-Paque isolated mononuclear cells

Reagents for esterase identification of monocytes on slide preparations are suitable for staining Ficoll-Paque isolated mononuclear cells in suspension. The suspension method is rapid and costless, and permits an accurate evaluation of esterase-positive cell percentages in mixed cell populations.

Establishment of long-term monocyte suspension cultures from normal human peripheral blood.

The long-term suspension growth of normal, immature myeloid cells from fresh human cord blood was recently reported and required cells separated on supplemented discontinuous Percoll gradients, growth in media containing hydrocortisone and vitamins D3, and gentle, continuous agitation (13). When normal adult bone marrow (six donors) or blood from Epstein-Barr virus (EBV)-seropositive donors (nine donors) was used as a source of fresh human leukocytes, only short-term proliferation of myeloid cells was achieved with the same techniques. However, when leukocytes prepared from EBV seronegative normal adult peripheral blood were used, pure populations of monocytes and macrophages that replicate slowly in liquid suspension culture for greater than 5 mo were repeatedly obtained from three independent donors. These cultures consists of several morphologically distinguishable monocytic cell types, including an approximately 20% adherent macrophage population. The monocytic nature of these cultures was confirmed by cytochemical, immunological, and functional criteria. These monocytes retain a normal chromosome pattern and can be induced to differentiate to phagocytic cells by treatment with tetradecanylphorbal acetate. Eventually, the cultures terminate as nonreplicating mature macrophages. These liquid suspension cultures should be a valuable resource for morphological, biochemical, and functional studies of developing monocyte-macrophages and their interaction with other cell types in normal and various pathological situations.

Cyclic nucleotides regulate the morphologic alterations required for chemotaxis in monocytes.

The initial morphologic response of human monocytes to chemoattractants is a change in shape from round to a triangular "motile" configuration (polarization). At doses chemotactic in vitro, chemoattractants induced rapid (t 1/2 = 45 sec), sustained (greater than 40 min) polarization of monocytes in suspension. Extracellular Ca++ was not required for polarization induced by chemoattractants, but in the absence of Ca++ kinetics were slowed (t 1/2 = 6.5 min). Phenylephrine, carbamycholine, serotonin, and ascorbate also caused rapid polarization of monocytes. Unlike chemoattractants, polarization by the pharmacologic agents was unsustained (less than 15 min), absolutely required extracellular Ca++, and affected about 50% of the cells responsive to chemoattractants. Based on relative sensitivities to alpha 1- and alpha 2-adrenergic agonists and antagonists, polarization caused by adrenergic agents was mediated by alpha 2-receptors. Muscarinic and alpha 2-adrenergic agonists, serotonin, and ascorbate enhanced the rate and number of monocytes polarizing to suboptimal doses of chemoattractants. Thus, the initial morphologic changes induced by chemoattractants appear to utilize an activation pathway shared with a variety of agents that enhance cGMP levels and inhibit adenylate cyclase. In contrast, theophylline, histamine, and isoproterenol, all agents that activate adenylate cyclase and elevate cAMP levels, inhibited monocyte polarization to chemoattractants. As in PMN, pharmacologic agents that increase cAMP levels inhibited monocyte chemotaxis in vitro, whereas those that inhibit adenylate cyclase and increase cGMP enhanced monocyte chemotactic responses. Thus, the initial morphologic response of monocytes to chemoattractants as well as the processes required for sustained directional motility are modulated by cyclic nucleotides.

Monoclonal antibody with specificity for monocytes and neurons

We have characterized a monoclonal antibody, called UC45, that reacts with both monocytes and neurons. It was derived from a fusion of the NS-1 plasmacytoma cell line with spleen cells from a mouse immunized with human acute monoblastic leukemia cells. The antibody reacts weakly with viable monocytes in suspension but has specificity for fibrous projections, which are found on monocytes that have adhered to a substrate. Other hemopoietically derived cells such as granulocytes and lymphocytes, and many tissue-culture lines, do not react with UC45 by cell-surface immunofluorescence. Similarly, UC45 reacts with the processes of both viable CNS and PNS neurons in tissue culture but with no other neural-tissue-derived cells. The monoclonal antibody has interspecies reactivity, in that it reacts with human, rat and mouse monocytes and neurons. The monocyte and neuronal antigen is present predominantly on a protein of 45 kd. Attempts to identify this protein on monocytes with conventional heteroantisera directed against fibronectin, complement components, fibrinogen, collagen, tubulin and actin have failed. A monoclonal antibody has therefore allowed identification of an antigen, unexpectedly shared by monocytes and neurons. The fact that it is found on cell processes of both cell types suggests that it may be performing some similar function for these cells, whose other activities differ substantially.

Tumor cell killing by freshly isolated peripheral blood monocytes

Human peripheral blood monocytes were reproducibly shown to lyse a variety of tumor cells in a 3- to 4-hr 51Cr release assay. Ficoll-Hypaque-purified mononuclear cells were suspended in medium supplemented with either 10% autologous serum or fetal calf serum (PCS). With either serum, highly purified (97–99%) and viable (>99%) monocyte suspensions were obtained by EDTA-reversible adherence to plastic surfaces which had been precoated with autologous serum. When used as effectors in cytotoxicity assays, the monocytes recovered from mononuclear cells suspended in FCS-supplemented medium exhibited higher cytolytic activity and were therefore used for further studies. Using FCS for both coating the plates and supplementing the suspension medium resulted in monocytes with low cytolytic activity. Tumor cell lysis measured by 51Cr release was detected within 2 hr of incubation and increased gradually with time. The level of lysis was dependent on the effector/target ratio and the tumor target cell employed. The involvement of natural killer lymphocytes in the observed tumoricidal activity was excluded. Detection of cytotoxic activity in a short-term assay will be very helpful in further studies of the mechanism of tumor cell killing by human monocytes since potential complicating effects of long-term in vitro cultivation will be minimized.

Antibody‐Dependent Monocyte‐Mediated Cytotoxicity.

Monocyte mediated lysis of human type B-erythrocytes coated with heat-inactivated anti-B serum was measured as 51Cr-release. Preincubation of monocytes with heat-inactivated normal AB serum inhibited cytolysis while a strong stimulation of cytolysis was seen with fresh AB serum. These effects of serum were reversible upon cell washing. Preincubation of monocytes with hyperimmune human serum against type B erythrocytes did not alter the cytolysis. After incubation at 37 degrees C of monocytes with immune complexes formed of human albumin and rabbit anti-human albumin a dose-related inhibition of cytolysis was observed. This inhibition was equally expressed at different albumin to anti-albumin ratios. Pretreatment with immune complexes at 4 degrees C had the same effect on cytolysis as pretreatment at 37 degrees C. The effect of immune complexes on monocyte-mediated cytolysis was resistant to repeated cell washing. After incubation of pretreated monocytes at 37 degrees C for up to 18 h some of the inhibition disappeared, but without normalization. Platelet depletion of monocyte suspensions resulted in a significant increase of cytolysis, and addition of washed platelets reinduced a decreased of cytolysis, which correlated with the number of platelets added.

Antibody‐Dependent Monocyte‐Mediated Cytotoxicity

Among mononuclear cells isolated from human blood monocytes exclusively mediated antibody‐dependent cytolysis of human type B erythrocytes. Adherent monocytes detached from plastic surfaces by lowering the temperature to 4°C expressed the same cytotoxicity as monocytes contained in the unseparated mononuclear cell fractions. No difference was observed in the cytotoxicity of non‐adherent monocytes and of monocytes remaining adherent at 4°C and subsequently mechanically removed. Removal of carbonyl‐iron phagocytosing monocytes reduced the number of latex‐phagocytosing monocytes without influencing cytotoxicity of the remaining monocytes. In vitro cultivation for 24h of unseparated mononuclear cells and of purified monocyte suspensions increased the number of latex‐positive cells, leaving the number of esterase‐positive cells and the cytotoxicity unchanged.

Effects of human neutrophils and monocytes on Nocardia asteroides: failure of killing despite occurrence of the oxidative metabolic burst.

To study mechanisms of resistance to Nocardia asteroides in vitro, suspensions of human neutrophils and monocytes were challenged with N. asteroides in the presence of autologous serum. At 4 hr, numbers of viable N. asteroides incubated with neutrophils and monocytes averaged only 10% and 21% less, respectively, than numbers of N. asteroides incubated without phagocytes. In contrast, a mean of > 90% of Listeria monocytogenes and Staphylococcus aureus incubated with neutrophils and monocytes was killed. The failure to kill N. asteroides was not associated with absence of an oxidative metabolic burst by the phagocytes, since chemiluminescence occurred when phagocytes encountered N. asteroides and was blocked in preparations of N. asteroides and neutrophils by preincubation of the neutrophils with chlorpromazine. Thus, when cultured in suspension, human neutrophils and monocytes are unable to kill significant numbers of N. asteroides despite the occurrence of an oxidative metabolic burst.

Relationship Between Monocytosis and T-Lymphocyte Function in Human Cancer2

This study was designed to: 1) determine the relative number of monocytes in mononuclear cell suspensions derived from the peripheral blood of cancer patients and 2) ascertain if any relationship existed between the numbers of monocytes in those cell suspensions and T-lymphocyte function. Monocytes were quantitated by morphology verified by phagocytosis of antibody-coated erythrocytes. A significant difference (P less than or equal to 0.01) existed between the number of monocytes in suspensions from normal individuals (26.2 +/- 4.3) and cancer patients (38.0 +/- 13.4). The cancer patients were divided into 2 groups: 1) those who exhibited normal in vitro T-cell responses to phytohemagglutinin and 2) those in whom responses were significantly suppressed. The mean number of monocytes in suspensions from the cancer patient group with normal responses was 29 +/- 9, whereas that from the cancer patients with suppressed responses was 47 +/- 11, a highly significant difference (P less than or equal to 0.01). Therefore, the study demonstrated two things: 1) Mononuclear cell suspensions derived from cancer patients exhibited significant monocytosis relative to those from normal individuals and 2) a strong correlation existed between monocytosis and suppressed T-lymphocyte function in vitro.

Pretreatment of plastic petri dishes with fetal calf serum. A simple method for macrophage isolation

We have developed a simple method which can recover the highly purified macrophages or monocytes in suspension from mouse peritoneal exudate cells and human peripheral blood mononuclear cells. Plastic Petri dishes coated overnight with heat-inactivated fetal calf serum (FCS) selectively bind macrophages and monocytes. The adherent macrophages and monocytes are easily removed by incubation in phosphate-buffered saline containing 0.2% ethylenediamine tetraacetate (EDTA) and 5% FCS, and recovered as a cell suspension with greater than 95% purity. A small number of isolated cells can restore the mitogenic response to phytohemagglutinin (PHA-P) of macrophage-depleted lymphocytes and can lyse 51Cr-labeled target cells in an antibody-dependent cell-mediated cytotoxicity system. Thus, the method should be valuable for studies of various functions of macrophages and monocytes from different immune tissues of man and animals.

Importance of HLA-D antigens for the cooperation between human monocytes and T lymphocytes.

AbstractThe ability of autologous and allogeneic monocytes to restore the responsiveness of purified T lymphocytes to tuberculin (PPD) was studied in twelve different experiments involving eleven unrelated T lymphocyte donors and a large number of related and unrelated monocyte donors. T lymphocyte suspensions were prepared by passage of peripheral blood mononuclear cell suspensions through Ig‐anti‐Ig‐coated columns. Monocyte‐enriched suspensions were prepared from mononuclear suspensions by density gradient centrifugation on albumin solution and added to the T cell suspension in a final concentration of 3%. Autologous monocytes and monocytes from related donors sharing one HLA haplotype with the T cell donor partly restored the responsiveness to PPD, and so did monocytes from unrelated donors who shared HLA‐D alleles with the T cells. None of eight monocyte suspensions from eight unrelated donors sharing no HLA‐D antigens with the T cell donor were able to restore the responsiveness to PPD. The difference between HLA‐D‐sharing and nonsharing monocytes could not be explained by a simultaneous allogeneic reaction, as the addition of monocytes autologous with the T cells to mixtures of non‐HLA‐D‐sharing T cells and monocytes partly restored the response. Moreover, experiments with the addition of PPD to ordinary mixed leukocyte cultures revealed only a moderate depression on the PPD response. It is concluded that in man – as in mice and guinea pigs – some HLA‐D identity between monocytes and T lymphocytes is necessary for a cooperation to take place in the secondary immune response. This observation emphasizes the homology between HLA‐D antigens in man and Ia antigens in animals.

Purification of Human Monocytes on Microexudatecoated Surfaces

Abstract Human blood monocytes, but not other Ficoll-Hypaque cells, adhere to plastic surfaces from which confluent BHK cells have been removed. EDTA (3 mM) rapidly and completely removes monocytes, providing a simple technique for preparation of human monocytes in suspension. The method should be valuable for studies of monocyte function where monocyte monolayers are not suitable.

Identification of Monocytes in Suspensions of Mononuclear Cells

A new histochemical technique for morphological studies of mononuclear cells and granulocytes, based on fluorescent staining with methyl green, pyronine Y, and stilbene-isothiocyanato disulfonic acid (MPS stain) is described. The method was applied to mononuclear cells isolated from the blood of normal human subjects by Ficoll-Isopaque density centrifugation. Three cell populations were distinguished, mainly on the basis of differences in morphology and cytochemistry, utilizing the MPS stain. One of the cell types had many of the morphological characteristics of the monocyte. This technique, augmented by lysosomal content and endocytosis capacity studies, revealed contimination of the cell suspension with a larger percentage of monocytes than has usually been reported in the literature.