Functional and phenotypic changes associated with the in vitro development of human monocytes into activated macrophages

Freshly isolated human peripheral blood monocytes had minimal cytotoxic effect in vitro on the schistosomula of Schistosoma mansoni. However, stimulation of the cells with either interferon gamma (IFN) or specific anti-parasite antiserum caused an increase in cytotoxicity. Additionally, the normal development of monocytes into macrophages over 7 days was associated with a sharp increase in cytotoxicity. The non-cytotoxic monocytes were compared with activated macrophages to assess whether cytotoxicity was associated with changes in immunophenotype. As monocytes developed into macrophages there were marked increases in transferrin receptors (HB21), macrophage cellular integrin (3.9), and Fc receptors (KB61). A further three markers showed increased expression in 7-day-old macrophages stimulated by IFN, namely a high affinity Fc gamma receptor (10.1), MHC Class II (1B5) and tumour necrosis factor (TNF).     

Antagonistic and additive effects of IL-4 and interferon-gamma on human monocytes and macrophages: effects on Fc receptors, HLA-D antigens, and superoxide production

During an immune response a multitude of lymphokines are produced which modulate the function of mononuclear phagocytes. In this study, we investigated possible additive, synergistic, or antagonistic effects of three lymphokines, IL-4 (1-100 U/ml, 0.01-1 ng/ml), interferon-gamma (IFN) (1-100 U/ml) and IL-2 (30-300 U/ml) on Fc receptors (FcR1 and FcR2), complement receptors (CR3 and CR4), and HLA-D antigens (HLA-DR and HLA-DQ) on human monocytes and macrophages. Exposure of monocytes to IL-4 alone resulted in changes in the expression of all these receptors. Both FcR1 and FcR2 were downregulated in a dose-dependent manner while the expression of CR3, CR4, HLA-DR, and HLA-DQ was increased. Antagonistic effects of IL-4 and IFN were observed on FcR1 and FcR2; IL-4-induced downregulation of the FcR1 and FcR2 was inhibited by IFN, and vice versa, IFN-induced upregulation of FcR1 and FcR2 was inhibited by IL-4. Phagocytosis of particulate immune complexes (EAs) as well as production of superoxide (O2-) in response to EAs were inhibited by IL-4, and the inhibition was reversed by IFN. Antagonistic effects of IL-4 and IFN were also observed on CR3 and CR4 expression. Additive effects of IL-4 and IFN were on the other hand seen on HLA-DR and HLA-DQ expression as well as on O2- production in response to stimulation with phorbol ester (PMA). The addition of IL-2 to IL-4 and/or IFN-containing cultures had no further modulatory effect on receptor expression or O2- production. In vitro matured macrophages (M phi) had a similar response pattern to IL-4 and IFN as the freshly isolated monocytes. Alveolar macrophages (AM phi), on the other hand, did not modulate FcR1 and HLA-DQ in response to IL-4, and downregulated FcR2 in response to IFN. Antagonistic effects of the two factors were only seen on CR expression. These results imply that FcR expression and function on monocytes and inflammatory macrophages may be in sensitive balance with the relative concentrations of IL-4 and IFN in the immune environment. FcRs on AM phi are less responsive to modulation by these lymphokines.     

Role of interferon γ and tumour necrosis factor α in monocyte-mediated cytostasis and cytotoxicity against a human histiocytic lymphoma cell line

Summary In view of cellular adoptive immunotherapy we have studied monocyte-mediated cytostasis and cytotoxicity against U 937 cells, a human histiocytic lymphoma cell line. Highly purified human monocytes and monocytederived macrophages were activated with interferon (IFN) or tumour necrosis factor (TNF) to antileukemic immune effector cells. Antileukemic activity of human monocytes was dependent on monocyte differentiation into macrophages and on a dose- and time-dependent activation with IFN or TNF. Maximum cytostasis of 97.0±0.7% (mean ± SEM) (conventional [³H]dT uptake assay) and 81.9±5.3% cytotoxicity (modified MTT assay) of U 937 cells was obtained by monocytes activated with 100 U/ml IFN for at least 24 h at an effector-to-target-cell ratio of 10. U 937 cells premodified with IFN showed an increase in susceptibility to monocyte-mediated cytotoxicity. U 937 cells premodified with TNF were almost resistant to monocyte-mediated cytotoxicity while activated monocytes maintained their cytotoxic potential. These data show that IFN and TNF are potent activators of monocyte-mediated cytotoxicity. Furthermore, IFN and TNF might be involved in the regulation of the susceptibility of leukemic cells to lysis by interactions with monocytes or macrophages.     

Expression of the lysophospholipid receptor family and investigation of lysophospholipid-mediated responses in human macrophages

Some of the biological effects of lipoproteins have been attributed to their association with lysophosphatidic acid (LPA), lysophosphatidylcholine (LPC), sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC). These lysophospholipids mediate multiple biological responses via several G protein-coupled receptors (GPR). The expression of these receptors, however, has not been systematically investigated in primary human monocytes and macrophages as major cells involved in atherosclerosis. The mRNAs for all 15 receptors described so far were detected in monocytes, macrophages, foam cells and high density lipoprotein (HDL(3))-treated cells using real time RT-PCR. Immunoblots revealed that S1P(1), S1P(2), S1P(4), LPA(1), LPA(2) and GPR65 are expressed in monocytes and macrophages, while S1P(5) and LPA(3) have not been detected. S1P(3) was induced during differentiation but down-regulated by lipid-loading and HDL(3), whereas LPA(1) was down-regulated in differentiated macrophages. The influence of S1P on macrophages was investigated and the induction of CD32 indicates an enhanced phagocytic activity. Altogether, these data give insights into the expression and regulation of lysophospholipid receptors in primary human monocytes, macrophages and foam cells.     

Macrophages modulate the viability and growth of human mesenchymal stem cells

Following myocardial infarction, tissue repair is mediated by the recruitment of monocytes and their subsequent differentiation into macrophages. Recent findings have revealed the dynamic changes in the presence of polarized macrophages with pro‐inflammatory (M1) and anti‐inflammatory (M2) properties during the early (acute) and late (chronic) stages of cardiac ischemia. Mesenchymal stem cells (MSCs) delivered into the injured myocardium as reparative cells are subjected to the effects of polarized macrophages and the inflammatory milieu. The present study investigated how cytokines and polarized macrophages associated with pro‐inflammatory (M1) and anti‐inflammatory (M2) responses affect the survival of MSCs. Human MSCs were studied using an in vitro platform with individual and combined M1 and M2 cytokines: IL‐1β, IL‐6, TNF‐α, and IFN‐γ (for M1), and IL‐10, TGF‐β1, TGF‐β3, and VEGF (for M2). In addition, polarization molecules (M1: LPS and IFN‐γ; M2: IL‐4 and IL‐13) and common chemokines (SDF‐1 and MCP‐1) found during inflammation were also studied. Indirect and direct co‐cultures were conducted using M1 and M2 polarized human THP‐1 monocytes. M2 macrophages and their associated cytokines supported the growth of hMSCs, while M1 macrophages and their associated cytokines inhibited the growth of hMSCs in vitro under certain conditions. These data imply that an anti‐inflammatory (M2) environment is more accommodating to the therapeutic hMSCs than a pro‐inflammatory (M1) environment at specific concentrations. J. Cell. Biochem. 114: 220–229, 2012. © 2012 Wiley Periodicals, Inc.     

Regulation of oxidative metabolism by interferon-gamma during human monocyte to macrophage differentiation

The capacity of human peripheral blood monocytes to generate highly reactive oxygen-derived molecules was studied during differentiation of the cells to macrophages in vitro. The effect of semipurified native interferon gamma (IFN gamma) on the differentiation-associated production of active oxygen intermediates was assessed by continuous exposure of the cells to IFN gamma or by adding it to the cultures at different stages of in vitro differentiation. Chemiluminescence (CL) response, triggered by opsonised zymosan, was highest in fresh isolated monocytes and fell constantly during a two-week culture. IFN gamma had little effect on CL. Generation of intracellular O2- was determined by the reduction of nitroblue tetrazolium (NBT). Zymosan-induced NBT reduction increased slightly during monocyte to macrophage differentiation and was further enhanced by continuous presence of IFN gamma. Hydrogen peroxide (H2O2) release, triggered by phorbol myristate acetate (PMA), was low in monocytes, increased slightly, reaching a maximum on day 3, and declined thereafter. H2O2 secretion was greatly enhanced by the presence of IFN gamma and remained raised for at least 14 d. When added at intervals to spontaneously matured monocytes, IFN gamma had only modest and transient effects on the generation of intracellular O2- and H2O2. It is concluded that IFN gamma seems so to modulate human mononuclear phagocyte differentiation that they maintain or increase their oxidative metabolic capacity.     

Bone morphogenetic protein 7 polarizes THP-1 cells into M2 macrophages

It was hypothesized that monocyte treatment with bone morphogenetic protein 7 (BMP7) would significantly enhance monocyte polarization into M2 macrophages as well as increasing the levels of anti-inflammatory cytokines. In a cell culture system using monocytes (human acute monocytic leukemia cell line THP-1), we studied the effects of BMP7 on monocytes polarizing into M2 macrophages. The data demonstrate that THP-1 cells contain a BMP type II receptor (BMPR2), and that its activation is significantly (p < 0.05) increased following treatment with BMP7. Furthermore, there was an increase of M2 macrophages, BMPR2, and anti-inflammatory cytokines interleukin (IL)-10 and IL-1ra compared with the respective controls. Moreover, treatment with BMP7 caused a significant (p < 0.05) decrease in the levels of pro-inflammatory cytokines IL-6, tumour necrosis factor (TNF-α), and monocyte chemotactic protein-1 (MCP-1), compared with the controls. In conclusion, we suggest for the first time that BMP7 has a unique potential to polarize monocytes into M2 macrophages, required for tissue repair, which will have significant applications for the treatment of atherosclerosis.     

Interferon affects the formation of adhesion plaques in human monocyte cultures

When monocytes isolated from human blood adhere to glass substratum, actin- and vinculin-containing punctate plaques rapidly appear at the ventral surface of the cells. We show here that highly purified human leukocyte interferon (IFN) can inhibit formation of these adhesion plaques in a dose-dependent manner. Complete inhibition was obtained when 300 IU/ml IFN were added into the cell-seeding medium. Plaques already formed in the absence of IFN were only partially affected by subsequent addition of IFN into the culture medium. Prevention by IFN of the formation of the adhesion plaques was associated with loosened attachment of the cells to the substratum. Effect of IFN on cellular morphology was complex. At higher doses, IFN added to the cultures within 24 h of seeding almost completely inhibited the differentiation of monocytes to macrophages and most of the cells remained rounded. At lower doses, however, an enhancement of the bipolar spreading was seen and the end result was a culture with predominantly elongated fibroblastoid cells. The latter cells, unlike the fibroblastoid cells in untreated monocyte-macrophage cultures, were completely devoid of the actin plaques, while the reorganization of vimentin-type intermediate filaments took place in a normal manner. These results further support the view that the actin- and vinculin-containing plaques have a role in mediating firm adherence of human monocytes to growth substratum.     

Expression of the transferrin receptor gene during the process of mononuclear phagocyte maturation

The expression of transferrin receptors by blood monocytes, human alveolar macrophages, and in vitro matured macrophages was evaluated by immunofluorescence, radioligand binding, and Northern analysis, using the monoclonal anti-human transferrin receptor antibody OKT9, [125I]-labeled human transferrin and a [32P]-labeled human transferrin receptor cDNA probe, respectively. By immunofluorescence, the majority of alveolar macrophages expressed transferrin receptors (86 +/- 3%). The radioligand binding assay demonstrated the affinity constant (Ka) of the alveolar macrophage transferrin receptor was 4.4 +/- 0.7 X 10(8) M-1, and the number of receptors per cell was 4.4 +/- 1.2 X 10(4). In marked contrast, transferrin receptors were not present on the surface or in the cytoplasm of blood monocytes, the precursors of the alveolar macrophages. However, when monocytes were cultured in vitro and allowed to mature, greater than 80% expressed transferrin receptors by day 6, and the receptors could be detected by day 3. Consistent with these observations, a transferrin receptor mRNA with a molecular size of 4.9 kb was demonstrated in alveolar macrophages and in vitro matured macrophages but not in blood monocytes. Thus, although blood monocytes do not express the transferrin receptor gene, it is expressed by mature macrophages, an event that probably occurs relatively early in the process of monocyte differentiation to macrophages.     

Interferon-induced inhibition of receptor-mediated endocytosis of colony-stimulating factor (CSF-1) by murine peritoneal exudate macrophages

Apart from its characteristic antiviral activity, interferon (IFN) also exerts a variety of biologic effects on macrophages. We have studied the effect of IFN on the expression of the colony-stimulating factor receptors (CSF-1 receptors) by murine peritoneal exudate macrophages (PEM). At 37 degrees C, murine IFN decreased the expression of the CSF-1 receptor activity in a time- and dose-dependent fashion by PEM from both endotoxin-sensitive (C3H/Sn) and endotoxin-resistant strains (C3H/HeJ) of mice. Scatchard analysis from the binding data suggests that the decreased expression of CSF-1 receptors is a result of decreased number of receptors rather than a decreased binding affinity. When IFN was incubated with anti-IFN before the addition to cultures, the effect was completely abolished indicating that this activity resides in the same molecules as IFN. The suppressed CSF-1 receptor activity on PEM by IFN appeared to be stable. Removal of added IFN never resulted in a full recovery of CSF-1 binding activity by PEM even after prolonged incubation (7 days). IFN also inhibited the receptor-mediated uptake and utilization of CSF-1 molecules by treated cells, which appeared to be a direct effect of the decreased number of CSF-1 receptors. Treatment of PEM with dexamethasone, prostaglandin, transferrin, insulin, or dibutyryl cAMP failed to suppress both the expression of CSF-1 receptors and CSF-1 utilization by PEM. These studies suggest that IFN may play a role in the regulation of both macrophage production and differentiation via the modulation of specific membrane receptors and inhibition of receptor-mediated CSF-1 endocytosis.     

Change in the chemiluminescence reactivity pattern during in vitro differentiation of human monocytes to macrophages

We determined the luminol-enhanced chemiluminescence (CL) of fresh human monocytes and monocytes cultured for 1-14 days in vitro, within hydrophobic membranes, using a variety of stimuli known to trigger the respiratory burst of phagocytes. It was assured that CL emerged from an adherent subpopulation of mononuclear cells; polymorphonuclear leukocytes (PMN) contaminating mononuclear leukocytes (MNL) contributed little, if anything, to the CL response of MNL. Typical response patterns were established for fresh monocytes triggered by phorbol 12-myristate 13-acetate (PMA), zymosan, the Ca2+ ionophore A 23187, antibody-coated erythrocytes and Sendai virus. Differentiation in vitro into macrophages was associated with a general decrease in magnitude of the CL peak, in an overproportional decrease of the A23187 triggered response and in a complete loss of the response to Sendai virus--a loss which could not be prevented by addition of myeloperoxidase (MPO). In contrast to monocyte CL, macrophage CL was resistant to sodium azide, indicating its MPO-independent origin. Macrophage-type reactivity was obtained at day 4 of culture. Activation of macrophages with recombinant interferon-gamma for the last 2 days of culture was associated with a quantitative (approx. threefold) increase of the CL signal, although qualitatively the same reactivity pattern was obtained as with control macrophages. In contrast to luminol-dependent CL, the lucigenin-dependent CL response of macrophages was greater than that of monocytes, an increase which was particularly prominent for PMA stimulation.     

Regulation of P2X(7) nucleotide receptor function in human monocytes by extracellular ions and receptor density

P2X receptors function as ATP-gated cation channels. The P2X(7) receptor subtype is distinguished from other P2X family members by a very low affinity for extracellular ATP (millimolar EC50) and its ability to trigger induction of nonselective pores on repeated or prolonged stimulation. Previous studies have indicated that certain P2X(7) receptor-positive cell types, such as human blood monocytes and murine thymocytes, lack this pore-forming response. In the present study we compared pore formation in response to P2X(7) receptor activation in human blood monocytes with that in macrophages derived from these monocytes by in vitro tissue culture. ATP induced nonselective pores in macrophages but not in freshly isolated monocytes when both cell types were identically stimulated in standard NaCl-based salines. However, ion substitution studies revealed that replacement of extracellular Na+ and Cl- with K+ and nonhalide anions strongly facilitated ATP-dependent pore formation in monocytes. These ionic conditions also resulted in increased agonist affinity, such that 30-100 microM ATP was sufficient for activation of nonselective pores by P2X(7) receptors. Comparison of P2X(7) receptor expression in blood monocytes with that in macrophages indicated no differences in steady-state receptor mRNA levels but significant increases (up to 10-fold) in the amount of immunoreactive P2X(7) receptor protein at the cell surface of macrophages. Thus ability of ATP to activate nonselective pores in cells that natively express P2X(7) receptors can be modulated by receptor subunit density at the cell surface and ambient levels of extracellular Na+ and Cl-. These mechanisms may prevent adventitious P2X(7) receptor activation in monocytes until these proinflammatory leukocytes migrate to extravascular sites of tissue damage.     

Human peritoneal macrophages possess two populations of IgG Fc receptors

To characterize the binding properties of the Fc receptors on human macrophages, the binding of radiolabeled human IgG1 to peritoneal macrophages was assessed. Cells were obtained at the time of diagnostic laparoscopy from women undergoing evaluation of infertility. Macrophages bound on the average more IgG1 monomer than monocytes but the avidity with which both types of cells bound IgG1 monomer was comparable. By contrast, macrophages bound much more IgG1 dimers than monocytes. Scatchard plots of the binding of dimer to monocytes were linear, but plots of binding to macrophages were markedly curvilinear. This curvilinearity was not an artifact of extensive ligand internalization or catabolism by cells, since 80% of binding was reversible and there was very little catabolism of ligand in the medium. Assuming that the observed curvilinearity was due to the presence of two independent subpopulations of receptors, an objective estimate for the number of receptors per cell and of the avidity with which each subpopulation bound IgG1 dimer was obtained using a previously described computer program (Scatfit). The analysis of the binding of dimer to macrophages from six donors suggested the presence of 42,000 +/- 33,000 high avidity receptors per cell which bind IgG1 dimer with a mean Ka of 2.7 X 10(9) M-1 and 218,000 +/- 127,000 low avidity receptors which bind the same ligand with a Ka of 1.1 X 10(7) M-1. ADCC of IgG antibody-coated sheep red blood cells mediated by macrophages was less readily inhibited by soluble IgG1 monomer than ADCC mediated by peripheral blood monocytes. This provides further evidence for the presence of low avidity receptors which bind monomeric IgG1 poorly and also suggests that these sites are functionally active in triggering antibody-dependent immune clearance.     

Phagocytic activity and protein content of human monocytes cultivated in the presence of various homologous interferon preparations

We have studied the effect of different types of interferons (IFN) on phagocytic activity and protein content when present during in vitro cultivation of human blood monocytes. Recombinant IFN-alpha and partially and highly purified leukocyte IFN preparations blocked the increase in phagocytic activity and protein content that occurs during in vitro cultivation of human monocytes. Fibroblast IFN blocked the increase in protein content, but did not significantly alter the phagocytic activity. IFN-gamma slightly enhanced phagocytic activity and protein content, while lymphoblastoid IFN preparations had no effect. The phagocytic activity and protein content of monocytes matured in vitro without IFN and then treated with IFN for 24 h was also tested. Phagocytosis via the non-specific receptors and the protein content was reduced by treatment of these cells with the IFN-alpha preparations. On the other hand Fc-receptor mediated phagocytosis was stimulated by IFN-gamma. Our data indicate that IFN effects on monocytes in culture varies depending on type and possibly subtypes of IFNs, and also on the timing of the treatment.     

Regulation of plasminogen receptor expression on human monocytes and monocytoid cell lines

The capacity of human monocytoid cell lines and peripheral blood monocytes to modulate their expression of plasminogen receptors has been assessed. After PMA stimulation, THP-1 or U937 monocytoid cells were separated into adherent and nonadherent populations. Plasminogen bound to adherent cells with similar capacity and affinity as to nonstimulated cells. In contrast, the nonadherent cells bound plasminogen with 5-17-fold higher capacity (without a change in affinity). This increase was selective as urokinase bound with similar affinity and capacity to the adherent and nonadherent populations. Upregulation of plasminogen receptors on the nonadherent monocytoid cells was rapid, detectable within 30 min, and reversible, adhesion of the nonadherent cells resulted in a sixfold decrease in plasminogen binding within 90 min. The increase in plasminogen binding to the nonadherent cells was associated with a marked increase in their capacity to generate plasmin activity from cell-bound plasminogen. PMA stimulation of human peripheral blood monocytes increased their expression of plasminogen receptors by two- to fourfold. This increase was observed in both adherent and nonadherent monocytes. Freshly isolated monocytes maximally bound 5.0 x 10(5) plasminogen molecules per cell, whereas monocytes cultured for 18 h or more maximally bound 1.7 x 10(7) molecules per cell, a 30-fold difference in receptor number. These results indicate that both monocytes and monocytoid cell lines can rapidly and markedly regulate their expression of plasminogen binding sites. As enhanced plasminogen binding is correlated with an increased capacity to generate plasmin, an enzyme with broad substrate recognition, modulation of plasminogen receptors may have profound functional consequences.     

Differential use of chondroitin sulfate to regulate hyaluronan binding by receptor CD44 in Inflammatory and Interleukin 4-activated Macrophages

CD44 is a cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is involved in processes ranging from leukocyte recruitment to wound healing. In the immune system, the binding of hyaluronan to CD44 is tightly regulated, and exposure of human peripheral blood monocytes to inflammatory stimuli increases CD44 expression and induces hyaluronan binding. Here we sought to understand how mouse macrophages regulate hyaluronan binding upon inflammatory and anti-inflammatory stimuli. Mouse bone marrow-derived macrophages stimulated with tumor necrosis factor α or lipopolysaccharide and interferon-γ (LPS/IFNγ) induced hyaluronan binding by up-regulating CD44 and down-regulating chondroitin sulfation on CD44. Hyaluronan binding was induced to a lesser extent in interleukin-4 (IL-4)-activated macrophages despite increased CD44 expression, and this was attributable to increased chondroitin sulfation on CD44, as treatment with β-d-xyloside to prevent chondroitin sulfate addition significantly enhanced hyaluronan binding. These changes in the chondroitin sulfation of CD44 were associated with changes in mRNA expression of two chondroitin sulfotransferases, CHST3 and CHST7, which were decreased in LPS/IFNγ-stimulated macrophages and increased in IL-4-stimulated macrophages. Thus, inflammatory and anti-inflammatory stimuli differentially regulate the chondroitin sulfation of CD44, which is a dynamic physiological regulator of hyaluronan binding by CD44 in mouse macrophages.