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PubMed1990

Evaluation of the antibody-dependent cytotoxic capabilities of individual human monocytes. Role of Fc gamma RI and Fc gamma RII and the effects of cytokines at the single cell level.

In this report we present evidence that not all human peripheral blood monocytes mediate antibody-dependent cellular cytotoxicity (ADCC), and that this function may be determined on an individual cell by both the type and level of expression of FcR, and by the state of cellular activation and/or differentiation. Although the diverse range of effector and regulatory functions performed by human monocytes suggests the possibility of distinct subsets, it is not clear whether observed functional heterogeneity reflects the presence of true monocyte subpopulations, or whether this diversity represents a continuum of maturational states present in the peripheral circulation. In an attempt to address this question, we investigated the ability of human monocytes to carry out ADCC at the single cell level, with emphasis on the role of the three FcR for IgG (Fc gamma RI, Fc gamma RII, and Fc gamma RIII) in mediating cytotoxicity. Using a modified plaque assay, 58.3% +/- 4.9 of freshly isolated monocytes mediated ADCC, as evidenced by the formation of lytic plaques in monolayers of ox erythrocyte (oxE) target cells. Significant increases in the number of plaque-forming cells were observed after positive selection by flow microfluorimetry for those monocytes expressing high levels of Fc gamma RI and Rc gamma RII, but not Fc gamma RIII. Bispecific antibodies composed of Fab fragments of anti-oxE antibody covalently coupled to Fab fragments of anti-Fc gamma R antibodies were used to independently evaluate the ability of Fc gamma RI, Fc gamma RII, and Fc gamma RIII to mediate single cell cytotoxicity. Significant increases in the number of plaque-forming cells were observed in the presence of anti-Fc gamma RI x anti-oxE and anti-Fc gamma RII x anti-oxE bispecific antibodies, confirming the efficiency of Fc gamma RI and Fc gamma RII as cytotoxic trigger molecules on human monocytes. Incubation of monocytes with purified rIFN-gamma and granulocyte macrophage-CSF, but not IL-2, IL-3, IL-4, IL-6, or TNF-alpha, also resulted in significant increases in the number of monocytes mediating cytotoxicity, suggesting that cytotoxic ability at the single cell level may be influenced by factors which effect monocyte activation and differentiation, respectively. Overall, these studies demonstrate that freshly isolated human monocytes are heterogeneous in their ability to mediate ADCC, and suggest that this functional diversity arises not from discrete subpopulations of cells, but from a continuum of maturational/activational states present within the peripheral circulation.

Antibody-Dependent Cell Cytotoxicity↗

PubMed1990

Monocyte-T-cell interactions in pokeweed mitogen-activated cultures.

Monocyte-T-cell interactions were studied in pokeweed mitogen (PWM)-activated cell cultures. We addressed the question of monocyte changes in PWM-stimulated cultures of T cells and monocytes and found, by flow cytometric analysis, that PWM activation led to a loss of cells with monocyte or macrophage phenotype (CD14, HLA-DR, HLA-DQ) within 48 hr of culture in the presence of T cells (CD4+ T cells), but not in cultures of pure monocytes. Chemiluminescence measurements revealed that phagocytic stimulation of monocytic superoxide release was impaired in PWM-stimulated cultures of monocytes plus T cells, but not in PWM-stimulated cultures of pure monocytes. Furthermore, PWM induced the secretion of interferon-gamma (IFN-gamma) in primary cultures of T cells supplemented with 20% of monocytes, whereas in subsequent secondary cultures of these cells PWM induced IFN-gamma only when monocytes were added. We conclude from these flow cytometric and functional analyses that monocytes are efficiently eliminated from PWM-activated T-cell/monocyte cultures by CD4+ T lymphocytes.

Antigens, Differentiation, Myelomonocytic↗

PubMed1992

Stimulus-response coupling in monocytes infected with Leishmania. Attenuation of calcium transients is related to defective agonist-induced accumulation of inositol phosphates.

Mononuclear phagocytes infected with Leishmania have been shown to have defective responses to extracellular stimuli. To investigate the potential relationship of these findings to alterations in calcium-dependent signaling pathways, the regulation of [Ca2+]i concentrations was examined in human peripheral blood monocytes infected with amastigotes of Leishmania donovani. Measurements of [Ca2+]i in fura-2-loaded monocytes were made at the single cell level by microfluorimetry. In normal monocytes, resting [Ca2+]i was 56 +/- 2 nM (mean +/- SEM). In contrast, in monocytes infected with Leishmania there was an approximately twofold increase in basal [Ca2+]i (122 +/- 5 nM, p less than 0.01 vs control). Treatment of cells with pertussis toxin before infection did not abrogate infection-induced increases in basal [Ca2+]i, suggesting that this effect was not mediated via the activation of a G protein coupled to phospholipase C. However, elevated resting [Ca2+]i did correlate with increased rates of 45Ca2+ uptake by infected monocytes. As expected, in response to treatment with 10(-7) M FMLP, control monocytes showed rapid net increases in [Ca2+]i of 303 +/- 19 nM. In contrast, net transients of [Ca2+]i in infected monocytes in response to FMLP were attenuated to only 137 +/- 9 nM (p less than 0.01 vs control). This result was not related to excess buffering of [Ca2+]i in infected cells as both control and infected monocytes showed equivalent transients of [Ca2+]i in response to the calcium ionophore A23187. Rather, inhibition of agonist-induced calcium release in infected cells appeared related to defective generation of second messenger because compared to control cells labeled with myo-[2-3H]inositol, little accumulation of inositol 1,4,5-trisphosphate was detected in infected monocytes. Attenuation of inositol phosphate accumulation and calcium release in response to chemotactic peptide correlated with decreased FMLP-induced superoxide and hydrogen peroxide production by infected monocytes. These results provide direct evidence for defective regulation of [Ca2+]i and calcium-dependent signaling in Leishmania-infected monocytes and provide a basis for understanding abnormalities in activation-related responses that involve signaling through Ca(2+)-regulated pathways.

Animals↗

PubMed1991

Suppression of human monocyte function against Candida albicans by autologous IL-2-induced lymphokine-activated killer cells.

We have previously reported that IL-2-induced lymphokine-activated killer (LAK) cells have the capacity to lyse autologous and allogeneic monocytes. To understand the biologic significance of this interaction, we investigated the function of human monocytes against the opportunistic pathogen, Candida albicans, subsequent to a short exposure to autologous LAK cells. A highly sensitive radiolabel assay, which makes use of the incorporation of [3H]glucose into residual Candida after their incubation with monocytes, was developed to measure antifungal activity. Cultured monocytes, after 2 to 6 h exposure to LAK cells, were found to be substantially suppressed in their ability to control fungal growth. Moreover, monocytes cultured in the presence of granulocyte/macrophage (GM)-CSF or IL-3, were even more suppressed in function after a short incubation with LAK cells. The effect of GM-CSF was both time and dose dependent, with peak susceptibility induced after 4 days of culture with as little as 10 U/ml of the cytokine. These GM-CSF-cultured monocytes, however, were relatively resistant to inhibition by freshly isolated large granular lymphocytic NK cells. Therefore, IL-2 induces in large granular lymphocytic cells the capacity to inhibit monocyte function. In contrast to GM-CSF and IL-3, IFN-gamma was found to have a protective effect on monocytes, because monocytes cultured 4 days in IFN-gamma were not significantly inhibited by LAK cells. These results indicate that LAK cells may be involved in regulation of monocyte function and suggest that the state of differentiation induced by different cytokines may dictate the level of control of the monocytes by LAK cells.

Candida albicans↗

PubMed1991

Down-regulation of surface FcRI and decrease in antibody-dependent cellular cytotoxicity of cultured monocytes. Reversal by monensin or cytochalasin-D.

To investigate the mechanisms involved in regulation of antibody-dependent cellular cytotoxicity (ADCC) mediated by human monocytes, 51Cr-labeled sheep red blood cells (RBC) were used as target cells in vitro. Monocytes incubated overnight at 37 degrees C before addition of SRBC and antibody exhibited a significant decrease in ADCC activity compared with freshly isolated cells. This pattern was observed with monocytes from all donors tested, regardless of the media used for culture. Supernatants from monocyte cultures did not inhibit the cytotoxic ability of fresh monocytes and cycloheximide, a protein synthesis inhibitor, could not reverse ADCC suppression in cultured monocytes, indicating that the alteration in ADCC is probably not due to inhibitory molecules secreted or synthesized during incubation. A correlation between the decrease in the number of surface FcRI and loss in ADCC ability of cultured monocytes was found. One mechanism for the reduced FcRI expression of 1-day-old monocytes may be rapid internalization that exceeds the rate of reexpression, because cytochalasin-D or monensin, each of which inhibits receptor internalization, maintained FcR expression as well as ADCC ability of cultured monocytes. These data illustrate mechanisms whereby alteration in the number of receptors may underlie loss of receptor-mediated functions, or be involved in augmentation of their biologic activity. The findings that important monocyte functions change under conditions of storage or culture have relevance to in vitro testing of various immune functions of monocytes performed clinically to monitor or guide therapy.

Animals↗

PubMed1990

Monocytes enhance the bidirectional release of type I plasminogen activator inhibitor by endothelial cells.

Human umbilical vein endothelial cells cultured on a collagen lattice were used to study the effects of the interaction between human monocytes and endothelial cells on the production of type 1 plasminogen activator inhibitor (PAI-1) by endothelial cells. The effects of adherence and transendothelial migration of monocytes on endothelial PAI-1 release were compared with those of other leukocytes, conditioned media from monocytes, and interleukin-1 beta (IL-1 beta). Because the cell culture system used allows simultaneous analysis of the lumenal and the subendothelial compartment of endothelial cell monolayers, we also studied into which direction PAI-1 is released by endothelial cells. Under quiescent conditions, the net amount of PAI-1 accumulated at the lumenal side was twofold higher than that accumulated at the subendothelial side (about 2.0 micrograms PAI-1/10(6) cells and 1.1 microgram PAI-1/10(6) cells, respectively, in 24 hours), as analyzed by a quantitative immunoradiometric assay (IRMA). Direct cell-cell contact between highly purified monocytes and endothelial cells strongly enhanced the PAI-1 release by endothelial cells in a dose-dependent way, whereas lymphocytes and neutrophils did not affect endothelial PAI-1 production. The monocyte-mediated increase was first detected after 12 hours of incubation and lasted for at least 48 hours. In the presence of two monocytes per endothelial cell, the increases of PAI-1 at the lumenal side and at the subendothelial side were 87% and 32% in 24 hours, respectively. The effect of IL-1 beta on PAI-1 release by endothelial cells closely resembled that observed for monocytes. Monocyte-conditioned medium contained heat-labile product(s) which also, although to a much lesser extent than intact monocytes, enhanced endothelial PAI-1 release. Similarly, monocytes cultured on top endothelial cell separated by a microporous filter enhanced the release of PAI-1 to a lesser extent. Thus, these findings indicate that monocytes enhance endothelial PAI-1 release by mechanisms that are, at least in part, dependent on cell-cell contact.

Cell Adhesion↗

PubMed1985

A beta-glucan inhibitable receptor on human monocytes: its identity with the phagocytic receptor for particulate activators of the alternative complement pathway.

The ligand specificity of the human monocyte receptor that mediates phagocytosis of particulate activators of the human alternative complement pathway was defined by inhibiting the phagocytic response with glycans known to be present in zymosan. When monocytes in monolayers were preincubated with 100 micrograms/ml of beta-glucan and then incubated with 1.25 to 2.5 X 10(6) zymosan particles, the percentage of cells that exhibited phagocytosis was inhibited in a time-dependent manner; maximal inhibition occurred within 20 min of preincubation. beta-Glucan inhibited monocyte phagocytosis of zymosan and rabbit erythrocytes (Er) in a similar dose-dependent fashion and at 100 micrograms/ml reduced monocyte ingestion of 5 X 10(6)/ml zymosan and 2 X 10(8)/ml Er by 63 +/- 8% and 68 +/- 16% (mean +/- SD, n = 3), respectively. The other glycan constituent of zymosan, mannan, was less than 1% as active, and 10 mg/ml of mannan reduced the number of monocytes ingesting zymosan and Er by 56 +/- 12% and 26 +/- 11%, respectively. At concentrations as high as 500 micrograms/ml, beta-glucan had no effect on monocyte Fc, C3b, or fibronectin receptor-mediated functions. Enzymatic hydrolysis of beta-glucan and alpha-mannan with beta-glucosidase or beta-glucanase before their incubation with monocytes abrogated their inhibitory capacity, whereas hydrolysis with alpha-mannosidase or alpha-glucosidase did not. Neither of the two alpha-glucans tested (dextran T-70 and nigeran) affected monocyte ingestion of zymosan particles or sheep erythrocytes (Es) sensitized with rabbit 7S anti-Es (EsIgG) at concentrations as high as 2 mg/ml. In contrast, a number of beta-glucans were active against zymosan but not EsIgG ingestion with a 75% reduction in the number of monocytes ingesting zymosan occurring with 100 micrograms/ml laminarin, 500 micrograms/ml soluble pachyman, and 900 micrograms/ml of soluble pustulan. The galactan, agarose, either in suspensions at 2 mg/ml or in a soluble portion at 600 micrograms/ml failed to affect monocyte ingestion of zymosan particles or Er. Thus, the monocyte receptor for particulate activators that is specifically inhibited by beta-glucan at a rate compatible with a phagocytic process and that recognizes beta-glucans but not alpha-glucans, mannan, or galactan is a beta-glucan receptor.

Binding, Competitive↗

PubMed1989

Induction of human monocyte susceptibility to lymphokine-activated killer cell lysis by granulocyte-macrophage colony-stimulating factor.

We have recently reported that cultured human monocytes are susceptible to lysis by autologous lymphokine-activated killer (LAK) cells. In an attempt to modulate the sensitivity of monocytes to LAK-mediated lysis, monocytes were cultured in the presence of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF was found to enhance the susceptibility of monocytes to lysis by LAK cells by 2- to 5-fold over that of untreated cells in a dose-dependent manner. As little as 10 units of GM-CSF per milliliter was sufficient to induce increased sensitivity. In a kinetics study, susceptibility of monocytes increased after 2 days of incubation with GM-CSF, with peak sensitivity occurring from 4 to 6 days of culture. The effect of GM-CSF appeared to be specific for monocytes within the circulating peripheral blood cells because nonadherent cells (NAC) and granulocytes, which are normally resistant to LAK-mediated lysis, did not become susceptible after treatment with GM-CSF. In cold-target inhibition experiments, unlabeled GM-CSF-treated monocytes, but not untreated monocytes, could block the lysis of FMEX, a human melanoma tumor cell line, as well as freshly isolated tumor cells. Finally, LAK cells specifically bound to GM-CSF-treated monocytes in significantly higher percentages than to control monocytes. In summary, our results indicate that GM-CSF was capable of enhancing the susceptibility of monocytes to LAK lysis possibly via increased binding or expression of target structure(s).

Cell Separation↗

PubMed1988

Phagocytosis of heat-killed blastospores of Candida albicans by human monocyte beta-glucan receptors.

Candida albicans is an opportunistic human pathogen with a cell wall that is qualitatively similar in carbohydrate composition to zymosan. Monolayers of human peripheral blood monocytes ingested 2.5 x 10(6)-2.5 x 10(7) heat-killed C. albicans blastospores in a dose-related manner. The percentage of monocytes ingesting at least one C. albicans reached a near plateau level of 68 +/- 6% (mean +/- SD, n = 3) when 2.5 x 10(7) particles per ml were incubated with monocytes for 30 min. Pretreatment of monocytes with 10 ng/ml to 100 micrograms/ml of soluble yeast beta-glucan inhibited C. albicans ingestion in a dose-dependent manner without affecting Fc-mediated ingestion of IgG-coated sheep erythrocytes (EsIgG). Pretreatment of monocytes with 100 micrograms/ml of soluble yeast beta-glucan inhibited the ingestion of 1 or more C. albicans by 80 +/- 11% (mean +/- SD, n = 4), with 50% inhibition occurring with approximately 7 micrograms/ml of soluble beta-glucan. Incubation of monocytes with 100 micrograms/ml to 1000 micrograms/ml of soluble yeast mannan resulted in no significant inhibition of C. albicans ingestion. The pretreatment of monolayers of monocytes for 30 min with 1 microgram/ml to 50 micrograms/ml of affinity-purified trypsin resulted in a dose-dependent inhibition of C. albicans ingestion with 10 micrograms/ml of trypsin inhibiting the percentage of monocytes ingesting 1 or more organisms by 71 +/- 6%. The inhibitory effects of soluble yeast beta-glucan and trypsin pretreatment on the ingestion of heat-killed C. albicans are comparable to the inhibitory effect of these agents on monocyte phagocytosis of zymosan and glucan particles. This relationship indicates that C. albicans contain a ligand that is recognized by the monocyte beta-glucan receptor of human monocytes.

Candida albicans↗

PubMed1985

Lysis of tumor cells by human blood monocytes by a mechanism independent of activation of the oxidative burst.

Tumorilytic human blood monocytes recognize and destroy neoplastic cells by a mechanism that is nonphagocytic and requires cell-to-cell contact. The mechanism of cytolysis subsequent to binding is controversial. Release of reactive oxygen intermediates by activated rodent macrophages has been suggested as an important mechanism for tumor cell lysis in some short-term cytotoxicity assays. We examined whether oxygen intermediates are also responsible for mediating the lysis of adherent human tumor cells in a long-term (72-h) tumoricidal assay. Human blood monocytes were incubated with medium, concanavalin A-stimulated lymphokine [macrophage-activating factor (MAF)], lipopolysaccharide endotoxin, or human recombinant gamma interferon for 24 h prior to the addition of [125I] iododeoxyuridine-labeled A375 melanoma cells. The following evidence indicated that monocyte-mediated tumor cell lysis was independent of superoxide anion (O2-) and H2O2 production: (a) although human blood monocytes incubated for 24 h with gamma interferon produced twice as much O2- as control or MAF-treated monocytes, gamma interferon did not activate monocyte tumoricidal activity unless combined with lipopolysaccharide endotoxin, 0.2 ng/ml or more; (b) incubating the monocytes with 10 nM phorbol myristate acetate for 0.5 h stimulated O2- production but no cytotoxicity; (c) the cytolytic activity of MAF-treated monocytes was not decreased in the presence of catalase or superoxide dismutase; and (d) finally, peripheral blood monocytes were isolated from six patients with chronic granulomatous disease, activated by MAF or lipopolysaccharide endotoxin, and then assayed for tumoricidal activity. While these activated chronic granulomatous disease monocytes did not produce O2- or H2O2, tumor cell lysis was normal in all six patients. Hence, lysis of tumor cells in a 72-h assay is not dependent upon the generation of O2- and/or H2O2 and is intact in chronic granulomatous disease monocytes.

Catalase↗

PubMed1986

Platelet glycoproteins IIb and IIIa associated with blood monocytes are derived from platelets.

Platelet glycoprotein IIb/IIIa (GP IIb/IIIa), the receptor complex for fibrinogen, has been regarded as a megakaryocyte/platelet lineage-restricted antigen. Recently, however, it has been reported that GP IIb/IIIa is expressed in blood monocytes. Studies were performed to establish the origin and immunological characteristics of monocyte-associated glycoproteins IIb and IIIa (GPs IIb and IIIa). Preparations of blood monocytes containing varying platelet-monocyte ratios were metabolically labeled with [35S]methionine with the expectation that any newly synthesized GPs IIb and IIIa would be monocyte-derived, since platelets have only rudimentary protein synthetic apparatuses. Analyses of sodium dodecyl sulfate (SDS) gels of homogenates of cell preparations containing from 200 to 5:1 platelet-monocyte ratios revealed that unlabeled GPs IIb and IIIa were readily immunoisolated using protein A-Sepharose immunobeads. However, fluorographic analyses of the same cell preparations pulse-labeled with [35S]methionine failed to demonstrate synthesis of GP IIb or IIIa. Additionally, no GP IIb or IIIa was detected when immunoisolation was carried out in pure preparations of monocytes containing less than 1:100 platelet-monocyte ratios and SDS acrylamide gels were stained by the sensitive silver stain method. Furthermore, heterologous polyspecific antisera and two monoclonal antibody preparations against GPs IIb and IIIa, which bound to platelets, failed to bind to monocyte membranes. Thus, evidence was presented that indicated that monocytes do not synthesize platelet GPs IIb and IIIa and that detection of these molecules in blood monocyte preparations reflects platelet contamination.

Animals↗

PubMed1986

Comparative analysis of the priming effect of human interferon-gamma, -alpha, and -beta on synergism with muramyl dipeptide analog for anti-tumor expression of human blood monocytes.

Freshly isolated human peripheral blood monocytes from healthy volunteers were not cytotoxic to allogeneic A375 melanoma cells, but they were activated to the cytotoxic state by incubation in vitro with either des-methyl muramyl dipeptide (norMDP; minimal effective dose, 0.5 micrograms/ml) or recombinant human interferon-gamma (rIFN-gamma; minimal effective dose, 1 U/ml). A combination of subthreshold concentrations of these agents (norMDP, 0.5 micrograms/ml; rIFN-gamma, 10 U/ml) also induced significant cytotoxicity, indicating that the effects of norMDP and rIFN-gamma in monocyte activation are synergistic. Natural human IFN-gamma (nIFN-gamma) and norMDP also had similar synergistic effects. Pretreatment of rIFN-gamma with anti-IFN-gamma antibody completely inhibited its synergistic effect with norMDP in monocyte activation. Because pretreatment of rIFN-gamma and norMDP with polymyxin B did not interfere with their effects in monocyte activation, the preparations were not contaminated with lipopolysaccharide. Moreover, because pretreatment of monocyte monolayers with anti-Leu-11b antibody (anti-natural killer (NK) cell antibody) and complement did not interfere with the synergistic effects of norMDP and rIFN-gamma, whereas pretreatment with anti-Leu-M1 antibody (anti-monocyte antibody) caused complete inhibition of their effects, the observed tumor cytotoxicity of monocyte-rich monolayers was probably not due to a small number of adherent NK cells, but to the stimulation of the monocytes. Natural and recombinant IFN-alpha and IFN-beta at concentrations of greater than or equal to 100 U/ml also induced tumoricidal activity of monocytes, but unlike IFN-gamma, their effects were additive with norMDP, and they had less priming effect than IFN-gamma when they were added before norMDP to monocytes. These findings suggest that recombinant human IFN-gamma has much more synergistic potential with norMDP than IFN-alpha or IFN-beta, and this synergism of rIFN-gamma and norMDP for monocyte activation could be of clinical value in treatment of disseminated malignant diseases, because these compounds are readily available at standardized concentrations.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1987

Tumor necrosis factor is an important mediator of tumor cell killing by human monocytes.

The mechanism of human peripheral blood monocyte-mediated cytotoxicity for tumor cells was investigated, using the A673 human rhabdomyosarcoma and HT-29 human colon adenocarcinoma lines as target cells. A673 cells were shown to be susceptible to the cytotoxic action of purified recombinant human tumor necrosis factor (TNF). A673 cells were also highly sensitive to the cytotoxic action of peripheral blood monocytes. Clones of A673 cells sensitive and resistant to TNF were isolated and characterized for their sensitivity to monocyte killing. A good correlation was found between the sensitivity of these clones to the cytotoxicity of TNF and their susceptibility to killing by monocytes. A TNF-specific neutralizing monoclonal antibody (MAb) reduced monocyte killing of parental A673 cells and of a TNF-sensitive clone of A673 cells. Inhibition of monocyte killing by this MAb was particularly pronounced at a low effector to target cell ratio. HT-29 cells were relatively resistant to the cytotoxic action of recombinant TNF and to monocyte killing. Treatment of HT-29 cells with recombinant human IFN-gamma increased their susceptibility to both TNF cytotoxicity and monocyte killing. In addition, MAb to TNF inhibited monocyte killing in HT-29 cells sensitized by incubation with IFN-gamma. Our data show that TNF is an important mediator of the cytotoxicity of human monocytes for tumor cells and that IFN-gamma can increase monocyte cytotoxicity by sensitizing target cells to the lytic action of TNF.

Adenocarcinoma↗

PubMed1988

Effect of recombinant granulocyte/macrophage colony-stimulating factor on human monocyte activity in vitro and following intravenous administration.

The purpose of these studies was to examine the antitumor properties of blood monocytes from patients undergoing a phase I trial with recombinant granulocyte/macrophage colony-stimulating factor (rGM-CSF). Peripheral blood monocytes from 7 patients receiving various doses of rGM-CSF by continuous infusion were isolated prior to therapy and at various times during the 2-wk infusion. Monocytes/cubic centimeter of blood increased in a dose-dependent fashion in patients receiving rGM-CSF. However, activation of monocyte-mediated tumorilytic properties was seen in only 1 of 7 patients. rGM-CSF administration also did not stimulate interleukin-1 or tumor necrosis factor production by blood monocytes. The failure to detect monocyte-mediated tumoricidal activation was not secondary to an inherent "defect" in the patients' monocytes because in vitro incubation with lipopolysaccharide alone or human recombinant gamma-interferon plus muramyl dipeptide resulted in monocyte-mediated cytotoxicity and in the secretion of interleukin-1 and tumor necrosis factor. rGM-CSF in vitro also did not stimulate the tumoricidal function of normal monocytes or the secretion of interleukin-1 or tumor necrosis factor. We concluded that systemic administration of rGM-CSF did not result in routine activation of monocyte-mediated cytotoxicity but did result in a dose-dependent rise in the number of circulating monocytes. Because these monocytes could be activated in vitro, we propose that, in an adjuvant therapy setting, rGM-CSF be combined with other activating agents to increase the pool of potential killer cells.

Cells, Cultured↗

PubMed1979

Phagocytic peripheral blood monocytes from rabbits and humans express membrane receptors specific for IgM molecules: evidence that incubation with neuraminidase exposes cryptic IgM (Fc) receptors.

Phagocytic human and rabbit peripheral blood monocytes, identified by their ingestion of polystyrene particles, were investigated for the presence of surface membrane receptors for IgM molecules. After incubation of freshly isolated monocytes with IgM anti-sheep erythrocyte (SRBC) preparations, a mean of 0.7% of human monocytes and a mean of 16.2% of rabbit monocytes formed rosettes with SRBC. However, if the monocytes were pre-incubated with vibrio cholerae neuraminidase (VCN), these figures increased to 32.6% and 37.8% respectively. The specificity of rosette formation by VCN-treated monocytes was established in several experiments; SRBC sensitized with a F(ab')2 preparation of an IgG anti-SRBC reagent completely failed to rosette with VCN-treated monocytes, and inclusion of IgM, but not other Ig or non-Ig protein molecules in the test medium, inhibited rosette formation. Further, and most important, rosette formation by human monocytes was inhibited by F(c)5mu but not by Fabmi fragments. These findings indicate that both rabbit and human monocytes express IgM-class specific membrane receptors for IgM molecules, that these receptors may be cryptic or hidden but can be revealed by treatment with VCN and that the human monocyte IgM receptor is F(c) specific. Further, the rabbit monocyte IgM receptor was shown to be trypsin-resistant.

Animals↗

PubMed1983

Pre-incubation of human monocytes results in loss of effector activity and diminished stimulation of the autologous mixed lymphocyte reaction.

Human monocytes were cultured at 37 degrees C for 72 h, washed, adjusted for viability and compared to freshly prepared monocytes for stimulation of the autologous mixed lymphocyte reaction (AMLR) and effector function. Pre-incubated monocytes were less potent AMLR stimulators than were freshly prepared cells. Pre-incubated monocytes demonstrated less antibody-dependent tumour killing of CCRF-CEM, less killing of Staphylococci and less spontaneous tumour killing of K-562 than did fresh monocytes. Pre-incubated monocytes produced less prostaglandin E2, demonstrated less surface Ia antigen and were less efficient accessory cells for antigen presentation than were fresh monocytes. AMLR stimulation correlated with monocyte killing (r = 0.95) and PGE2 production (r = 0.98). Thus, monocytes pre-incubated for 3 days are less active effector cells, display less surface Ia antigen and are less potent stimulators of the AMLR than fresh monocytes. Moreover, in this system, monocyte effector activity correlates with ability to stimulate the AMLR.

Antibody-Dependent Cell Cytotoxicity↗

PubMed1983

Characteristics and requirements of the interaction between human monocytes and tumor cells on the single cell level.

Highly purified (97%-99%) and viable (99%) peripheral blood monocytes obtained by EDTA-reversible adherence to autologous-serum-precoated plastic surfaces could rapidly lyse a variety of tumor cells in a 3-4 hr 51Cr release assay. Using these monocytes as effectors, a short-term agarose/conjugate assay was utilized, permitting us to examine the interaction between fresh human monocytes and neoplastic target cells on a single cell level. That the tumor-bound effector cells were indeed monocytes was confirmed by employing the monocyte-specific monoclonal antibody 61D3, which stained 95%-99% of the mononuclear cells bound to conjugated and killed K562 tumor targets. The binding of monocytes to target cells appeared to be temperature dependent and was extremely rapid, reaching a plateau after 5 min at 30 degrees C. Our findings demonstrated for the first time that only a proportion of human blood monocytes can bind to a particular target cell and that only a fraction of the binding cells have the intrinsic potential to kill those neoplastic targets. The proportion of monocytes capable of binding and killing varies between individuals and also depends on the tumor cell used, indicating heterogeneity in the monocyte and tumor cell populations. The highest proportion of monocytes bind to the human erythromyeloid leukemia K562 cell line (13%-50%). The frequency of monocytes capable of killing K562 tumor cells is relatively low (7%-13%). The system described here should be useful to study the heterogeneity of mononuclear phagocytes and to analyze the molecular basis of the interaction between those effector cells and neoplastic target cells.

Antibodies, Monoclonal↗

PubMed1984

Interferons as modulators of human monocyte-macrophage differentiation. I. Interferon-gamma increases HLA-DR expression and inhibits phagocytosis of zymosan.

The development of HLA-DR (Ia) expression in the presence and absence of interferon-gamma was monitored in monocyte-macrophage cultures. Overnight incubation with doses as low as 5 U/ml gave elevated values for Ia expression and the maximum increase was obtained with 200 U/ml. In contrast interferon-alpha had only a slight effect on the expression of Ia at doses as high as 2000 U/ml. The increase seen at 24 hr was maintained during the first 2 days of culture. The interferon-gamma-treated cells expressed four to five times more Ia than fresh monocytes. During the same time, monocytes cultured in the absence of interferon expressed approximately two times the amount of fresh monocytes. When the surface density of Ia was calculated, the interferon-gamma-treated monocytes expressed twice that of the untreated cells. Major changes in morphology and size occurred between days 3 and 4 of monocyte to macrophage development. Consequently a rapid increase in Ia expression took place; however, when the surface density was calculated this value increased only slightly when the monocytes matured to macrophages. The interferon-gamma-treated cells continued to express more total Ia as well as having increased surface density of this antigen. Interferon-gamma was also added to monocyte-macrophages several days after culture initiation (days 3, 4, and 5). Despite being in different stages of maturation, the cells responded to the interferon with increased Ia expression and surface density. The phagocytic activity of opsonized zymosans was also monitored. In contrast to Ia expression, this activity was downregulated by interferon-gamma, and the lower levels of phagocytosis were maintained through the 7 days of observation. Thus, interferon-gamma appears to change the differentiation pathway of the monocyte. The signal stimulates an increased level of Ia that may assist in the initiation of immune responses, and at the same time downregulates the scavenger role of removing opsonized particles. Once the monocyte has received this specific signal it continues to develop in a pathway different from that of the nontreated monocytes.

Cell Differentiation↗