Culture supernatants of different colon cancer cell lines induce specific phenotype switching and functional alteration of THP-1 cells

We developed an in vitro model to evaluate the effect of products secreted from different colorectal cancer (CRC) cell lines on specific phenotypic switching and functional alterations in THP-1 cells. We co-cultured the human monocytic cell line, THP-1, or phorbol-12-myristate-13-acetate (PMA)-treated THP-1 cells, (THP-1p), with supernatants from either the HT-29 (Dukes’ B), HCT-15 (Dukes’ C), or Colo205 (Dukes’ D) cell lines, and assessed the cells for macrophage differentiation. The surface marker and cytokine profiles suggested that secreted CRC factors differentiated THP-1 cells into a “mixed” M1/M2 phenotype, although HT-29 and Colo205 supernatants induced THP-1p cells into predominantly M1-like macrophages and M2-like macrophages, respectively. Further, all three CRC supernatants enhanced the phagocytic capacity and migration of THP-1 and THP-1p cells, altering their phenotype to a more M2-kind. Therefore, different CRC cell lines induced specific phenotype switching and functional polarization of THP-1 cells.     

Characterization and modeling of monocyte-derived macrophages after spinal cord injury

Spinal cord injury (SCI) elicits a neuroinflammatory reaction dominated by microglia and monocyte-derived macrophages (MDM). Because MDM do not infiltrate the spinal cord until days after injury, it may be possible to control whether they differentiate into neuroprotective or neurotoxic effector cells. However, doing so will require better understanding of the factors controlling MDM differentiation and activation. Our goal was to develop an in vitro model of MDM that is relevant in the context of SCI. This tool would allow future studies to define mechanisms and intracellular signaling pathways that are associated with MDM-mediated neuroprotection or neurotoxicity. We first characterized SCI-induced cytokine expression in MDM using laser capture microdissection and real-time PCR. Based on this data, we assessed which easily procurable primary macrophage subset would mimic this phenotype in vitro. We established the baseline and inductive potential of resident peritoneal, thioglycollate-elicited peritoneal and bone marrow-derived macrophages (BMDM) at the molecular, cellular and functional level. Of these cells, only BMDM retained the phenotypic, molecular and functional characteristics of MDM that infiltrate the injured spinal cord. Thus, peripheral macrophages should not be used interchangeably in vitro to model the functional consequences of the MDM response elicited by SCI.     

DcR3 protects THP-1 macrophages from apoptosis by increasing integrin α4

In order to analyze the function of DcR3 for the regulation of cell adhesion and apoptosis in macrophages, we investigated the expression of decoy receptor 3 (DcR3) in THP-1 monocytes/macrophages.DcR3 was expressed in THP-1 and increased by phorbol 12-myristate 13-acetate (PMA). The formation of macrophage aggregates was observed when THP-1 cells were differentiated by PMA or stimulated with DcR3-Fc. Undifferentiated THP-1 cells were also induced to form aggregates by DcR3-Fc. The expression of integrin α4 was significantly increased by DcR3-Fc. CHX-induced apoptosis in THP-1 was inhibited by DcR3-Fc, of which inhibition against CHX-induced apoptosis and aggregate formation were ameliorated by anti-VLA4 antibody.DcR3 may play a significant role in macrophages not only by a decoy receptor but also by increasing α4 integrin.     

Visceral Adipose MicroRNA 223 Is Upregulated in Human and Murine Obesity and Modulates the Inflammatory Phenotype of Macrophages

Obesity in humans and mice is typified by an activated macrophage phenotype in the visceral adipose tissue (VAT) leading to increased macrophage-mediated inflammation. microRNAs (miRNAs) play an important role in regulating inflammatory pathways in macrophages, and in this study we compared miRNA expression in the VAT of insulin resistant morbidly obese humans to a non-obese cohort with normal glucose tolerance. miR-223-3p was found to be significantly upregulated in the whole omental tissue RNA of 12 human subjects, as were 8 additional miRNAs. We then confirmed that miR-223 upregulation was specific to the stromal vascular cells of human VAT, and found that miR-223 levels were unchanged in adipocytes and circulating monocytes of the non-obese and obese. miR-223 ablation increased basal / unstimulated TLR4 and STAT3 expression and LPS-stimulated TLR4, STAT3, and NOS2 expression in primary macrophages. Conversely, miR-223 mimics decreased TLR4 expression in primary macrophage, at the same time it negatively regulated FBXW7 expression, a well described suppressor of Toll-like receptor 4 (TLR4) signaling. We concluded that the abundance of miR-223 in macrophages significantly modulates macrophage phenotype / activation state and response to stimuli via effects on the TLR4/FBXW7 axis.     

Oxygen Tension Modulates Differentiation and Primary Macrophage Functions in the Human Monocytic THP-1 Cell Line

The human THP-1 cell line is widely used as an in vitro model system for studying macrophage differentiation and function. Conventional culture conditions for these cells consist of ambient oxygen pressure (∼20% v/v) and medium supplemented with the thiol 2-mercaptoethanol (2-ME) and serum. In consideration of the redox activities of O₂ and 2-ME, and the extensive experimental evidence supporting a role for reactive oxygen species (ROS) in the differentiation and function of macrophages, we addressed the question of whether culturing THP-1 cells under a more physiologically relevant oxygen tension (5% O₂) in the absence of 2-ME and serum would alter THP-1 cell physiology. Comparisons of cultures maintained in 18% O₂ versus 5% O₂ indicated that reducing oxygen tension had no effect on the proliferation of undifferentiated THP-1 cells. However, decreasing the oxygen tension to 5% O₂ significantly increased the rate of phorbol ester-induced differentiation of THP-1 cells into macrophage-like cells as well as the metabolic activity of both undifferentiated and PMA-differentiated THP-1 cells. Removal of both 2-ME and serum from the medium decreased the proliferation of undifferentiated THP-1 cells but increased metabolic activity and the rate of differentiation under either oxygen tension. In differentiated THP-1 cells, lowering the oxygen tension to 5% O₂ decreased phagocytic activity, the constitutive release of β-hexosaminidase and LPS-induced NF-κB activation but enhanced LPS-stimulated release of cytokines. Collectively, these data demonstrate that oxygen tension influences THP-1 cell differentiation and primary macrophage functions, and suggest that culturing these cells under tightly regulated oxygen tension in the absence of exogenous reducing agent and serum is likely to provide a physiologically relevant baseline from which to study the role of the local redox environment in regulating THP-1 cell physiology.     

The peroxisome proliferator activated receptor δ is required for the differentiation of THP-1 monocytic cells by phorbol ester

BACKGROUND: PPARdelta (NR1C2) promotes lipid accumulation in human macrophages in vitro and has been implicated in the response of macrophages to vLDL. We have investigated the role of PPARdelta in PMA-stimulated macrophage differentiation.The THP-1 monocytic cell line which displays macrophage like differentiation in response to phorbol esters was used as a model system. We manipulated the response to PMA using a potent synthetic agonist of PPARdelta, compound F. THP-1 sub-lines that either over-expressed PPARdelta protein, or expressed PPARdelta anti-sense RNA were generated. We then explored the effects of these genetic modulations on the differentiation process. RESULTS: The PPARdelta agonist, compound F, stimulated differentiation in the presence of sub-nanomolar concentrations of phorbol ester. Several markers of differentiation were induced by compound F in a synergistic fashion with phorbol ester, including CD68 and IL8. Over-expression of PPARdelta also sensitised THP-1 cells to phorbol ester and correspondingly, inhibition of PPARdelta by anti-sense RNA completely abolished this response. CONCLUSIONS: These data collectively demonstrate that PPARdelta plays a fundamental role in mediating a subset of cellular effects of phorbol ester and supports observations from mouse knockout models that PPARdelta is involved in macrophage-mediated inflammatory responses.     

Adiponectin induces TNF-alpha and IL-6 in macrophages and promotes tolerance to itself and other pro-inflammatory stimuli

Adiponectin exerts anti-inflammatory effects via macrophages, suppressing the production of pro-inflammatory cytokines in response to bacterial lipopolysaccharide (LPS). Here, we provide experimental evidence that the "anti-inflammatory" effect of adiponectin may be due to an induction of macrophage tolerance: globular adiponectin (gAd) is a powerful inducer of TNF-alpha and IL-6 secretion in primary human peripheral macrophages, in the THP-1 human macrophage cell line, and in primary mouse peritoneal macrophages. Pre-exposure of macrophages to 10 microg/ml gAd rendered them tolerant to further gAd exposure or to other pro-inflammatory stimuli such as TLR3 ligand polyI:C and TLR4 ligand LPS, while pre-exposure to 1 microg/ml of and re-exposure to 10 microg/ml gAd unmasked its pro-inflammatory properties. GAd induced NF-kappaB activation and tolerance to further gAd or LPS exposure. Our data suggest that adiponectin constant presence in the circulation in high levels (in lean subjects) renders macrophages resistant to pro-inflammatory stimuli, including its own.     

即早基因c-fos在THP-1单核巨噬细胞亚型极化中的差异表达*

目的:探讨即早基因c-fos在THP-1巨噬细胞亚型极化过程中的表达变化。方法:运用PMA刺激诱导THP-1单核细胞极化为巨噬细胞,观察c-fos在单核细胞极化过程中的表达变化;在PMA刺激的基础上,分别运用LPS和IL-4诱导THP-1巨噬细胞向M1及M2亚型极化,实时定量PCR及Western blot技术分析刺激24 h时,细胞亚型标记物CD274、CD86和CD163的表达变化,并动态观察诱导极化过程中,c-fos的表达情况。结果:c-fos在PMA刺激THP-1单核细胞分化为巨噬细胞过程中蛋白和mRNA水平显示上调;LPS诱导THP-1巨噬细胞极化为M1型过程中,c-fos蛋白和mRNA水平表达降低,其特异性标记物在24 h呈现出M1型极化的特点(CD86蛋白表达升高,CD274、CD163蛋白表达降低);IL-4诱导THP-1巨噬细胞极化为M2型过程中,c-fos蛋白和mRNA水平表达升高,其特异性标记物在24 h表现出M2型极化的特点(CD86蛋白表达降低,CD274、CD163蛋白表达升高)。结论:c-fos参与了THP-1单核细胞向巨噬细胞极化的过程,并且可能通过抑制巨噬细胞M1亚型形成,促进巨噬细胞向M2亚型极化的作用参与巨噬细胞的亚型极化及其功能调节中。     

Lipid droplet: A functionally active organelle in monocyte to macrophage differentiation and its inflammatory properties

Lipid droplets (LDs) perform several important functions like inflammatory responses, membrane trafficking, acts as secondary messengers, etc. rather than simply working as an energy reservoir. LDs have been implicated as a controlling factor in the progression of atherosclerosis followed by foam cell formation that derives from macrophages during the differentiation process. However, the role of LDs in monocyte differentiation or its further immunological function is still an area that mandates in-depth investigation. We report that LD dynamics is important for differentiation of monocytes and is absolutely required for sustained and prolonged functional activity of differentiated macrophages. In THP-1 cell line model system, we elucidated that increase in total LD content in monocyte by external lipid supplements, can induce monocyte differentiation independent of classical stimuli, PMA. Differential expression of PLIN2 and ATGL during the event, together with abrogation of de novo lipogenesis further confirmed the fact. Besides, an increase in LD content by free fatty acid supplement was able to exert a synergistic effect with PMA on differentiation and phagocytic activity compared to when they are used alone. Additionally, we have shown Rab5a to play a vital role in LDs biosynthesis/maturation in monocytes and thereby directly affecting differentiation of monocytes into macrophages via AKT pathway. Thus our study reveals the multi-faceted function of LDs during the process of monocyte to macrophage differentiation and thereby helping to maintain the functional activity.     

TLR4-mediated survival of macrophages is MyD88 dependent and requires TNF-alpha autocrine signalling

Modulation of macrophage survival is a critical factor in the resolution of inflammatory responses. Exposure to LPS protects innate immune cells against apoptosis, although the precise pathways responsible for prolongation of macrophage survival remain to be fully established. The goal of this study was to characterize the mechanism of TLR4-mediated survival of murine bone marrow-derived macrophages upon M-CSF withdrawal in more detail. Using a combination of knockout mice and pharmacological inhibitors allowed us to show that TLR4 and TLR2 stimulation promotes long-term survival of macrophages in a MyD88-, PI3K-, ERK-, and NF-kappaB-dependent manner. LPS-induced long-term, but not short-term, survival requires autocrine signaling via TNF-alpha and is facilitated by a general cytoprotective program, similar to that mediated by M-CSF. TLR4-mediated macrophage survival is accompanied by a remarkable up-regulation of specific cell surface markers, suggesting that LPS stimulation leads to the differentiation of macrophages toward a mixed macrophage/dendritic cell-like phenotype.     

Morphine modulates monocyte-macrophage conversion phase

Monocyte migration and their activation into the macrophage phenotype play a role in the modulation of tissue injury. We studied the effect of morphine on the monocyte-macrophage conversion phase (MMCP). Phorbol 12-myristate 13-acetate (PMA) activated THP-1 cells and promoted their adhesion to the substrate. Morphine inhibited PMA-induced MMCP. However, opiate receptor antagonists attenuated this effect of morphine. Interestingly, PMA as well as morphine-stimulated superoxide production by monocytes. Superoxide dismutase (SOD) not only inhibited PMA-mediated MMCP but also attenuated the inhibitory effect of morphine. PMA not only enhanced adhesion of monocytes to a filter but also promoted their migration. These findings suggest that the PMA-induced macrophage phenotype conversion may be accelerating their migration; whereas, morphine may be preventing the migration of monocytes by inhibiting MMCP.     

α2-Macroglobulin synthesis by the human monocytic cell line THP-1 is differentiation state-dependent

Human α₂-macroglobulin (α₂M) is a broad spectrum proteinase inhibitor and cytokine carrier synthesized by a number of cell types including monocytes and macrophages. In this study, we report on the expression of α₂M by THP-1 cells. This monocytic cell line can be differentiated into a macrophage-like phenotype by treatment with interferon-γ (IFN-γ) or phorbol 12-myristate 13-acetate (PMA). α₂M was synthesized by THP-1 cells at a rate of 75 ng/10⁶ cells/24 h, as determined by Western blot analysis. After treating the cells with 500 U/ml of IFN-γ or with 100 ng/ml PMA, the synthesis rate increased to 219 ng/10⁶ cells/24 h and to 179 ng/10⁶ cells/24 h, respectively. The same agents also increased α₂M expression, as determined by Northern blot analysis. When the α₂M receptor antagonist, receptor associated protein (RAP), was included in the THP-1 medium, the amount of α₂M recovered in the conditioned medium increased. This result suggests that THP-1-secreted proteinases react with secreted α₂M and that the resulting complexes are catabolized by the α₂M receptor, which is also called low density lipoprotein receptor-related protein (LRP). We conclude that α₂M synthesis by THP-1 cells depends on the state of cellular differentiation. Reaction of α₂M with secreted proteinases may have minimized previous estimates of the rate of synthesis of α₂M by certain cells in culture. J. Cell. Biochem. 67:492–497, 1997. © 1997 Wiley-Liss, Inc.     

Plasticity of Human THP–1 Cell Phagocytic Activity during Macrophagic Differentiation

Studies of the role of macrophages in phagocytosis are of great theoretical and practical importance for understanding how these cells are involved in the organism’s defense response and in the development of various pathologies. Here we investigated phagocytic plasticity of THP–1 (acute monocytic human leukemia) cells at different stages (days 1, 3, and 7) of phorbol ester (PMA)–induced macrophage differentiation. Analysis of cytokine profiles showed that PMA at a concentration of 100 nM induced development of the proinflammatory macrophage population. The functional activity of macrophages was assessed on days 3 and 7 of differentiation using unlabeled latex beads and latex beads conjugated with ligands (gelatin, mannan, and IgG Fc fragment) that bind to the corresponding specific receptors. The general phagocytic activity increased significantly (1.5–2.0–fold) in the course of differentiation; phagocytosis occurred mostly through the Fc receptors, as shown previously for M1 macrophages. On day 7, the levels of phagocytosis of gelatin-and Fc–covered beads were high; however, the intensity of ingestion of mannan–conjugated beads via mannose receptors increased 2.5–3.0–fold as well, which indicated formation of cells with an alternative phenotype similar to that of M2 macrophages. Thus, the type and the plasticity of phagocytic activity at certain stages of macrophage differentiation can be associated with the formation of functionally mature morphological phenotype. This allows macrophages to exhibit their phagocytic potential in response to specific ligands. These data are of fundamental importance and can be used to develop therapeutic methods for correcting the M1/M2 macrophage ratio in an organism.     

Influence of biodegradable and non-biodegradable material surfaces on the differentiation of human monocyte-derived macrophages

Monocyte-derived macrophages (MDM) and multinucleated foreign body giant cells (FBGC) are the primary cell types that remain at the cell-material interface of polyurethane (PU)-based medical devices as a result of chronic inflammatory responses. In vitro studies have demonstrated that MDM possess degradative potential toward PU, which can result in device failure. Because most studies have followed the degradation potential, morphology, and function of these cells only once fully differentiated, the current study investigated the influence of a non-degradable control tissue culture-grade polystyrene (TCPS) surface relative to two degradable model polycarbonate-urethanes (PCNU), of different chemistry, on various parameters of MDM morphology and function during a 14-day differentiation time course. The differentiation of human monocytes isolated from whole blood on PCNU materials resulted in increased cell attachment, decreased multinucleation, and significant decreases in cell spreading when compared with cells differentiated on TCPS. Actin-stained podosome-like cell adhesion structures were increased in PCNU-adherent cells, accompanied by an alteration in beta-actin and vinculin protein expression. The expression of the CD68 macrophage marker was reduced when cells were adherent to the PCNU materials and compared with TCPS, suggesting altered cell activation by the degradable relative to non-degradable materials. The degradative potential of these cells was altered by the material surface they were exposed to as measured by esterase activity and protein expression of monocyte-specific esterase. This was also supported by physical material breakdown evident in scanning electron microscopy images that illustrated holes in the PCNU films generated by the presence of differentiating MDM. It was concluded from these studies that PCNU materials significantly alter the function and morphology of differentiating MDM. This must be taken into consideration when studying cell-material interactions because these cells will receive cues from their immediate environment (including the biomaterial) upon differentiation, thereby affecting their resulting phenotype.     

Activation of p38 MAPKalpha by extracellular pressure mediates the stimulation of macrophage phagocytosis by pressure

We have previously demonstrated that constant 20 mmHg extracellular pressure increases serum-opsonized latex bead phagocytosis by phorbol 12-myristate 13-acetate (PMA)- differentiated THP-1 macrophages in part by inhibiting focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK). Because p38 MAPK is activated by physical forces in other cells, we hypothesized that modulation of p38 MAPK might also contribute to the stimulation of macrophage phagocytosis by pressure. We studied phagocytosis in PMA-differentiated THP-1 macrophages, primary human monocytes, and human monocyte-derived macrophages (MDM). p38 MAPK activation was inhibited using SB-203580 or by p38 MAPK small interfering RNA (siRNA). Pressure increased phagocytosis in primary monocytes and MDM as in THP-1 cells. Increased extracellular pressure for 30 min increased phosphorylated p38 MAPK by 46.4 ± 20.5% in DMSO-treated THP-1 macrophages and by 20.9 ± 9% in primary monocytes (P < 0.05 each). SB-203580 (20 µM) reduced basal p38 MAPK phosphorylation by 34.7 ± 2.1% in THP-1 macrophages and prevented pressure activation of p38. p38 MAPK siRNA reduced total p38 MAPK protein by 50–60%. Neither SB-203580 in THP-1 cells and peripheral monocytes nor p38 MAPK siRNA in THP-1 cells affected basal phagocytosis, but each abolished pressure-stimulated phagocytosis. SB-203580 did not affect basal or pressure-reduced FAK activation in THP-1 macrophages, but significantly attenuated the reduction in ERK phosphorylation associated with pressure. p38 MAPK siRNA reduced total FAK protein by 40–50%, and total ERK by 10–15%, but increased phosphorylated ERK 1.4 ± 0.1-fold. p38 MAPK siRNA transfection did not affect the inhibition of FAK-Y397 phosphorylation by pressure but prevented inhibition of ERK phosphorylation. Changes in extracellular pressure during infection or inflammation regulate macrophage phagocytosis by a FAK-dependent inverse effect on p38 MAPK that might subsequently downregulate ERK. force; inflammation; infection; leukocyte; mechanotransduction; signal transduction