CD147 overexpression on synoviocytes in rheumatoid arthritis enhances matrix metalloproteinase production and invasiveness of synoviocytes

Macrophage-like synoviocytes and fibroblast-like synoviocytes (FLS) are known as the most active cells of rheumatoid arthritis (RA) and are close to the articular cartilage in a position enabling them to invade the cartilage. Macrophage-like synoviocytes and FLS expression of matrix metalloproteinases (MMPs) and their interaction has aroused great interest. The present article studied the expression of CD147, also called extracellular matrix metalloproteinase inducer, on monocytes/macrophages and FLS from RA patients and its potential role in enhancing MMPs and the invasiveness of synoviocytes. Expression of CD147 on FLS derived from RA patients and from osteoarthritis patients, and expression of CD147 on monocytes/macrophages from rheumatic synovial fluid and healthy peripheral blood were analyzed by flow cytometry. The levels of CD147, MMP-2 and MMP-9 mRNA in FLS were detected by RT-PCR. The role of CD147 in MMP production and the cells' invasiveness in vitro were studied by the co-culture of FLS with the human THP-1 cell line or monocytes/macrophages, by gel zymography and by invasion assay. The results showed that the expression of CD147 was higher on RA FLS than on osteoarthritis FLS and was higher on monocytes/macrophages from rheumatic synovial fluid than on monocytes/macrophages from healthy peripheral blood. RT-PCR showed that the expressions of CD147, MMP-2 and MMP-9 mRNA was higher in RA FLS than in osteoarthritis FLS. A significantly elevated secretion and activation of MMP-2 and MMP-9 were observed in RA FLS co-cultured with differentiated THP-1 cells or RA synovial monocytes/macrophages, compared with those co-cultured with undifferentiated THP-1 cells or healthy control peripheral blood monocytes. Invasion assays showed an increased number of invading cells in the co-cultured RA FLS with differentiated THP-1 cells or RA synovial monocytes/macrophages. CD147 antagonistic peptide inhibited the MMP production and the invasive potential. Our studies demonstrated that the CD147 overexpression on monocytes/macrophages and FLS in RA patients may be responsible for the enhanced MMP secretion and activation and for the invasiveness of synoviocytes. These findings suggest that CD147 may be one of the important factors in progressive joint destruction of RA and that CD147 may be a potential therapeutic target in RA treatment.     

Oxidized LDL induces the expression of ALBP/aP2 mRNA and protein in human THP-1 macrophages

The adipocyte lipid-binding protein (ALBP/aP2) belongs to a multigene family of fatty acid and retinoid transport proteins. This protein is abundantly expressed in the cytoplasm and nuclear region of adipocytes and is postulated to serve as a lipid shuttle, solubilizing hydrophobic fatty acids and delivering them to the appropriate metabolic system for utilization. This report demonstrates that human cholesterol-loaded THP-1 macrophages express ALBP/aP2 and that its expression can be stimulated by oxidized low density lipoprotein (oxLDL). The increase in mRNA expression was paralleled by a similar increase in ALBP/aP2 protein. The increase in ALBP/aP2 mRNA and protein in oxLDL-stimulated THP-1 macrophages is concentration and time dependent and is inhibited by treatment of the cells with an antioxidant inhibitor of nuclear factor-kappaB (NF-kappaB), pyrrolidine dithiocarbamate (PDTC), and with protein kinase C (PKC) inhibitors bisindolylmaleimide I and Ro-31-8220.These results suggest that activation of both NF-kappaB and PKC signaling pathways is necessary for oxLDL-induced ALBP/aP2 gene expression in THP-1 macrophages and that the upregulation of the fatty acid carrier may be a necessary event in foam cell formation.     

Th2 cell membrane factors in association with IL-4 enhance matrix metalloproteinase-1 (MMP-1) while decreasing MMP-9 production by granulocyte-macrophage colony-stimulating factor-differentiated human monocytes

Monocytes/macrophages are directly involved in tissue remodeling and tissue destruction through the release of matrix metalloproteinases (MMP). In the present study, we examined the effect mediated by contact of polarized Th cells with mononuclear phagocytes on the production of MMP-1, MMP-9, and their inhibitor. Plasma cell membranes from Ag-activated Th1 and Th2 cells were potent inducers of MMP-1 production by THP-1 cells. Cell membrane-associated TNF was found to be only partially involved in MMP-1 induction by both Th1 and Th2 cells. In Th2 cells exclusively, membrane-associated IL-4 induced MMP-1 production by THP-1 cells. This membrane-associated IL-4 effect was additive to that of TNF and was specifically observed on MMP-1 as MMP-9 production was concomitantly inhibited. Similarly, soluble IL-4 induced THP-1 cells to produce MMP-1, its effect proving additive to that of soluble TNF and to that of cell membranes of mitogen-activated HUT-78 cells. Its activity was blocked by IL-4 neutralization, and was unaffected by the presence of indomethacin. These effects on THP-1 cells were observed at protein and mRNA levels. Although inhibitory on freshly isolated peripheral blood monocytes, soluble IL-4 enhanced T cell-induced MMP-1 and inhibited MMP-9 production both at protein and mRNA levels in monocytes cultured for 7 days in the presence of GM-CSF. Thus, in contrast with previously reported effects, Th2 and IL-4 specifically induce MMP-1 production by mononuclear phagocytes at various stages of differentiation. This IL-4 activity may be relevant to pathological conditions dominated by Th2 inflammatory responses, resulting in tissue remodeling and destruction.     

Long noncoding RNA THRIL promotes foam cell formation and inflammation in macrophages

Tumor necrosis factor-α (TNF-α) and heterogenous nuclear ribonucleoprotein L (hnRNPL)-related immunoregulatory lincRNA (THRIL) is a long noncoding RNA (lncRNA) involved in various inflammatory diseases. However, its role in atherosclerosis is not known. In this study, we aimed to investigate the function of THRIL in mediating macrophage inflammation and foam cell formation. The expression of THRIL was quantified in THP-1 macrophages after treatment with oxidized low-density lipoprotein (oxLDL). The effect of THRIL overexpression and knockdown on oxLDL-induced inflammatory responses and lipid accumulation was determined. THRIL-associated protein partners were identified by RNA pull-down and RNA immunoprecipitation assays. We show that THRIL is upregulated in macrophages after oxLDL treatment. Knockdown of THRIL blocks oxLDL-induced expression of interleukin-1β (IL-1β), IL-6, and TNF-α and lipid accumulation. Conversely, ectopic expression of THRIL enhances inflammatory gene expression and lipid deposition in oxLDL-treated macrophages. Moreover, THRIL depletion increases cholesterol efflux from macrophages and the expression of ATP-binding cassette transporter (ABC) A1 and ABCG1. FOXO1 is identified as a protein partner of THRIL and promotes macrophage inflammation and lipid accumulation. Furthermore, overexpression of FOXO1 restores lipid accumulation and inflammatory cytokine production in THRIL-depleted macrophages. In conclusion, our data suggest a model where THRIL interacts with FOXO1 to promote macrophage inflammation and foam cell formation. THRIL may represent a therapeutic target for atherosclerosis.     

Troglitazone but not conjugated linoleic acid reduces gene expression and activity of matrix-metalloproteinases-2 and -9 in PMA-differentiated THP-1 macrophages

Gene expression and activity of matrix-metalloproteinases (MMP)-2 and -9 in macrophages are reduced through peroxisome proliferator-activated receptor gamma (PPARgamma)-dependent inhibition of NF-kappaB. Since conjugated linoleic acids (CLAs) are PPARgamma ligands and known to inhibit NF-kappaB via PPARgamma, we studied whether CLA isomers are capable of reducing gene expression and gelatinolytic activity of MMP-2 and -9 in PMA-differentiated THP-1 macrophages, which has not yet been investigated. Incubation of PMA-differentiated THP-1 cells with either c9t11-CLA, t10c12-CLA or linoleic acid (LA), as a reference fatty acid, resulted in a significant incorporation of the respective fatty acids into total cell lipids relative to control cells (P<.05). Treatment of PMA-differentiated THP-1 cells with 10 and 20 mumol/L troglitazone but not with 10 or 100 mumol/L c9t11-CLA, t10c12-CLA or LA reduced relative mRNA concentrations and activity of MMP-2 and MMP-9 compared to control cells (P<.05). DNA-binding activity of NF-kappaB and PPARgamma and mRNA expression of the NF-kappaB target gene cPLA(2) were not influenced by treatment with CLA. In contrast, treatment of PMA-differentiated THP-1 cells with troglitazone significantly increased transactivation of PPARgamma and decreased DNA-binding activity of NF-kappaB and relative mRNA concentration of cPLA(2) relative to control cells (P<.05). In conclusion, the present study revealed that CLA isomers, in contrast to troglitazone, did not reduce gene expression and activity of MMP-2 and -9 in PMA-differentiated THP-1 macrophages, which is probably explained by the observation that CLA isomers neither activated PPARgamma nor reduced DNA-binding activity of NF-kappaB. This suggests that CLA isomers are ineffective in MMP-associated extracellular matrix degradation which is thought to contribute to the progression and rupture of advanced atherosclerotic plaques.     

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。     

Expression of CD147 on phorbol-12-myris-tate-13-acetate (PMA)-treated U937 cells differentiating into foam cells

Monocytes, macrophages, and foam cells expressing CD147 can stimulate the production of several matrix metalloproteinases (MMPs) associated with the development of atherosclerosis. We defined the CD147 expression profile and examined the correlation between foam cell development and MMP-2, -9 expressions. Foam cells were derived from U937-stimulated macrophages using various concentrations of oxidized low-density lipoprotein (ox-LDL). PMA-stimulated U937 cells had a 4- to 5-fold increase in CD147 mRNA compared to undifferentiated monocytes and membrane-associated (mCD147) on foam cells decreased in response to ox-LDL in a dose-dependent manner compared to untreated macrophages. In contrast, ox-LDL treatment increased the levels of soluble CD147 (sCD147) and MMP-2, -9 in a dose-dependent manner. Our data suggested that monocyte differentiation up-regulated CD147 expression and lipid enrichment of foam cells had no effect on CD147 mRNA expression. Lipid loading in macrophages reduced mCD147 expression while increasing the levels of MMP-2, -9 and sCD147 in supernatants.     

Adiponectin induces CCL20 expression synergistically with IL-6 and TNF-α in THP-1 macrophages

Adiponectin (Ad) is an adipokine secreted from adipocytes. It is reported that Ad has many biological activities. However, its influence on inflammation is controversial. In the present study, we examined the influence of Ad on production of CCL20 from THP-1 macrophages. THP-1 macrophages were prepared from THP-1 monocytes by PMA treatment. THP-1 macrophages were cultured for 24h with Ad, IL-6, or TNF-α alone or with combinations of Ad and cytokines. CCL20 mRNA expression was then determined by real-time PCR. Full-length Ad (fAd) slightly but significantly induced CCL20 mRNA expression, and interestingly, co-stimulation with fAd and IL-6 or with fAd and TNF-α synergistically increased the expression of CCL20 mRNA. We explored the mechanism behind the synergistic effect of fAd and these cytokines. fAd did not affect the expression of receptors for IL-6 and TNF, and IL-6 and TNF-α did not increase the expression of the receptor for Ad in THP-1 macrophages. The increased expression of CCL20 by fAd is much higher in THP-1 macrophages compared with THP-1 monocytes. Furthermore, MMP-12 production was increased by IL-6 and TNF-α in THP-1 macrophages but it was not detectable in THP-1 monocytes. Treatment of fAd with MMP-12 induced globular Ad (gAd), and the expression of CCL20 in THP-1 macrophages was increased more potently by gAd than by fAd. MMP inhibitor (UK370106) inhibited the expression of CCL20 induced by co-stimulation with fAd and IL-6 or TNF-α. In conclusion, gAd played an important role in CCL20 expression, and MMP-12 induced by IL-6 or TNF-α was involved in the synergistic effect of fAd and cytokines.     

Hormone sensitive lipase mRNA in both monocyte and macrophage forms of the human THP-1 cell line

The identity of the neutral cholesteryl ester hydrolase (CEH) in human monocyte/macrophages is uncertain. Prior studies indicate that hormone sensitive lipase (HSL) is a major CEH in mouse macrophages, and that HSL mRNA is present in human THP-1 monocytes. In the present study, HSL mRNA expression was examined in THP-1 cells as a function of differentiation status and cholesterol enrichment. By RT-PCR with primer pairs that span exon boundaries, HSL mRNA was demonstrated in THP-1 monocytes and phorbol-ester differentiated THP-1 macrophages. cDNA identities were confirmed by sequencing. By Northern blotting, with HSL cDNA as probe, THP-1 monocytes were found to contain HSL mRNA of approximately 3 and 3.9 kb. In THP-1 macrophages, the 3 kb mRNA was greatly diminished, while the level of the 3.9 kb mRNA was maintained. mRNA of approximately 3 and 3.9 kb are those expected of the 86-kDa (adipocyte) and 117-kDa (testicular) HSL isoforms, respectively. The presence of the testicular isoform mRNA was confirmed in THP-1 cells by amplification and sequencing of an isoform-specific cDNA. Additionally, Northern-blot comparisons showed that the 3 and 3.9 kb mRNA in THP-1 comigrated with the HSL mRNA in 3T3-L1 adipocytes and rat testis, respectively. The level of the 3.9 kb mRNA did not vary greatly with cholesterol enrichment. Thus, the HSL gene is transcribed in THP-1 cells both before and after differentiation into macrophages; after differentiation, the predominant mRNA is that for the 117-kDa isoform. This isoform is a CEH, and may mediate some CE turnover in THP-1 cells.     

Cardiovascular drugs inhibit MMP-9 activity from human THP-1 macrophages

It is now recognized that atherosclerosis complications are related to the unstable character of the plaque rather than its volume. Vulnerable plaques often contain a large lipid core, a reduced content of smooth muscle cells, and accumulation of inflammatory cells. Colocalization of macrophages and active matrix metalloproteinases (MMPs) is likely relevant for atherosclerotic lesion disruption. Nevertheless, MMP activity and regulation by cardiovascular drugs remains poorly defined. In this study, we evaluated the effects of avasimibe, fluvastatin, and peroxisome proliferator-activated receptor (PPAR) ligands on 92-kDa gelatinase B (MMP-9) secretion by human THP-1 macrophages. THP-1 macrophages were treated with compounds for 48 h, and secreted MMP-9 protein was quantified by immunoassay. Avasimibe, fluvastatin, and PPARalpha agonists (fenofibric acid and Wy-14643) significantly reduced, in a concentration-dependent manner, MMP-9 protein (up to 67 +/- 5% for fenofibric acid). In these assays, the PPARgamma selective agonist rosiglitazone displayed a lower efficacy than other compounds. Enzymatic activity of MMP-9 was also decreased by all cardiovascular drugs tested. MMP-9 protein/activity inhibition by cardiovascular drugs was due, at least in part, to a decrease in MMP-9 mRNA. These results show that THP-1 macrophages could be an useful cellular model to investigate effects of compounds on plaque vulnerability through MMP-9 activity.     

Cytosolic,Autocrine Alpha-1 Proteinase Inhibitor (A1PI) Inhibits Caspase-1 and Blocks IL-1β Dependent Cytokine Release in Monocytes

Rationale

Activation state-dependent secretion of alpha-1 proteinase inhibitor (A1PI) by monocytes and macrophages was first reported in 1985. Since then, monocytes and tissue macrophages have emerged as key sentinels of infection and tissue damage via activation of self-assembling pattern recognition receptors (inflammasomes), which trigger inflammation and cell death in a caspase-1 dependent process. These studies examine the relationship between A1PI expression in primary monocytes and monocytic cell lines, and inflammatory cytokine expression in response to inflammasome directed stimuli.

Methods

IL-1 β expression was examined in lung macrophages expressing wild type A1PI (A1PI-M) or disease-associated Z isoform A1PI (A1PI-Z). Inflammatory cytokine release was evaluated in THP-1 monocytic cells or THP-1 cells lacking the inflammasome adaptor ASC, transfected with expression vectors encoding A1PI-M or A1PI-Z. A1PI-M was localized within monocytes by immunoprecipitation in hypotonic cell fractions. Cell-free titration of A1PI-M was performed against recombinant active caspase-1 in vitro.

Results

IL-1 β expression was elevated in lung macrophages expressing A1PI-Z. Overexpression of A1PI-M in THP-1 monocytes reduced secretion of IL-1β and TNF-α. In contrast, overexpression of A1PI-Z enhanced IL-1β and TNF- α secretion in an ASC dependent manner. A1PI-Z-enhanced cytokine release was inhibited by a small molecule caspase-1 inhibitor but not by high levels of exogenous wtA1PI. Cytosolic localization of A1PI-M in monocytes was not diminished with microtubule-inhibiting agents. A1PI-M co-localized with caspase-1 in gel-filtered cytoplasmic THP-1 preparations, and was co-immunoprecipitated with caspase 1 from nigericin-stimulated THP-1 cell lysate. Plasma-derived A1PI inhibited recombinant caspase-1 mediated conversion of a peptide substrate in a dose dependent manner.

Conclusions

Our results suggest that monocyte/macrophage-expressed A1PI-M antagonizes IL-1β secretion possibly via caspase-1 inhibition, a function which disease-associated A1PI-Z may lack. Therapeutic approaches which limit inflammasome responses in patients with A1PI deficiency, in combination with A1PI augmentation, may provide additional respiratory tissue-sparing benefits.     

Surfactant protein A increases matrix metalloproteinase-9 production by THP-1 cells

Matrix metalloproteinase (MMP)-9 from alveolar macrophages is a major source of elastolytic activity in the lung. It is increased in the bronchoalveolar lavage fluid of patients with emphysema. Although the importance of macrophage-derived elastolytic activity in the pathogenesis of emphysema is well established, questions remain about MMP-9 regulation and activity. Because surfactant protein A (SP-A) is capable of modulating other functions of human monocytic cells, we hypothesized that SP-A may regulate MMP-9 expression. Vitamin D3-differentiated THP-1 cells and peripheral blood mononuclear cells were stimulated in vitro with several concentrations of SP-A for different incubation times. MMP-9 mRNA expression was measured by dot-blot analysis, gelatinolytic activity in the medium was determined by gel zymography, protein expression was determined by ELISA, and a specific MMP-9 activity assay was used to measure the state of activation of this enzyme in the cell supernatants. SP-A induced the expression of MMP-9 in both cell types, the effect was time and dose dependent, and MMP-9 was released in its zymogen form. On the basis of results of neutralizing antibody studies, we believe that SP-A action is mediated through Toll-like receptor-2. Even though the biological meaning of these findings remains to be elucidated, these observations suggest the presence of a novel, locally controlled mechanism by which MMP-9 levels may be regulated in alveolar macrophages. We speculate that SP-A may influence the protease/antiprotease balance in the lungs of patients with quantitative and/or qualitative changes in surfactant constituents favoring an abnormal breakdown of extracellular matrix components.     

Expression of CD147 on monocytes/macrophages in rheumatoid arthritis: its potential role in monocyte accumulation and matrix metalloproteinase production

Monocytes/macrophages play an important role in rheumatoid arthritis (RA) pathogenesis. They can activate fibroblasts through many molecules, including IL-1 and tumor necrosis factor-alpha, but there have been very few reports on the role of CD147 in RA. In our study, the results of flow cytometry reveal that the mean fluorescence intensity (MFI) of CD147 expression on CD14+ monocytes of peripheral blood from RA patients was higher than that in normal control and ankylosing spondylitis (AS) patients. The MFI of CD147 expression on the CD14+ monocytes in RA synovial fluid was higher than that in RA peripheral blood. Immunohistochemical staining shows that CD147 expression in RA synovium correlated with matrix metalloproteinase (MMP)-1 expression. A double immunofluorescent assay shows that CD147 was expressed on CD68+ cells in RA synovium. The potential role of CD147 in cyclophilin A (CyPA)-mediated cell migration was studied using a chemotaxis assay in vitro and it was found that the addition of anti-CD147 antibody or a CD147 antagonistic peptide significantly decreased the chemotactic index of the mononuclear cells. The role of CD147 in MMP production and cell invasion in vitro were studied through the co-culture of human CD14+ monocytes or monocytic line THP-1 cells and human fibroblasts, as well as by gel zymography and an invasion assay. Significantly elevated release and activation of MMP-9 and/or MMP-2 were seen in the co-culture of human monocytes/THP-1 cells and fibroblasts compared with cultures of the cells alone. An increased number of cells invading through the filters in the invasion assays was also observed in the co-cultured cells. The addition of CD147 antagonistic peptide had some inhibitory effect, not only on MMP production but also on cell invasion in the co-culture. Our study demonstrates that the increased expression of CD147 on monocytes/macrophages in RA may be responsible for elevated MMP secretion, cell invasion and CyPA-mediated cell migration into the joints, all of which may contribute to the cartilage and bone destruction of RA. These findings, together with a better understanding of CD147, CyPA and RA, will help in the development of innovative therapeutic interventions for RA.     

Low-frequency electromagnetic fields do not alter responses of inflammatory genes and proteins in human monocytes and immune cell lines

The effects of low frequency electromagnetic fields (LF EMF) on human health are the subject of on-going research and serious public concern. These fields potentially elicit small effects that have been proposed to have consequences, either positive or negative, for biological systems. To reveal potentially weak but biologically relevant effects, we chose to extensively examine exposure of immune cells to two different signals, namely a complex multiple waveform field, and a 50 Hz sine wave. These immune cells are highly responsive and, in vivo, modulation of cytokine expression responses can result in systemic health effects. Using time course experiments, we determined kinetics of cytokine and other inflammation-related genes in a human monocytic leukemia cell line, THP-1, and primary monocytes and macrophages. Moreover, cytokine protein levels in THP-1 monocytes were determined. Exposure to either of the two signals did not result in a significant effect on gene and protein expression in the studied immune cells. Also, additional experiments using non-immune cells showed no effects of the signals on cytokine gene expression. We therefore conclude that these LF EMF exposure conditions are not expected to significantly modulate innate immune signaling.     

Differential regulation of MMP-1/9 and TIMP-1 secretion in human monocytic cells in response to Mycobacterium tuberculosis.

In tuberculosis, matrix metalloproteinase (MMP) secretion is involved in leukocyte migration to sites of infection but in excess may contribute to tissue destruction. We demonstrate that human monocytic THP-1 cells and primary monocytes secrete MMP-1 (52 kD collagenase) when phagocytosing live, virulent M. tuberculosis but not inert latex. The magnitude of MMP-1 secretion was approximately 10-fold less when compared to MMP-9 (92 kD gelatinase) secretion. MMP-1 secretion was also relatively delayed (detected at 24 h vs. 4 h). M. tuberculosis, zymosan or latex stimulate similar TIMP-1 secretion within 8 h and increasing over 24 h. MMP-1/9 secretion was decreased by inhibitors of protein kinase (PK) C, PKA or tyrosine kinases (PTK) in a concentration-dependent manner. In contrast, TIMP-1 secretion was not affected by PKC or PTK blockade and only somewhat reduced by high level PKA inhibition. In summary, M. tuberculosis-infected monocytes secrete MMP-1 at lower concentrations than MMP-9 and such MMP secretion is regulated by multiple upstream signalling pathways which do not control TIMP-1 secretion. Divergent effects of i on MMP and TIMP secretion from monocytes may be important in influencing matrix degradation in vivo.     

Triglyceride enhances susceptibility to TNF-α-induced cell death in THP-1 cells

Monocytes and macrophages play a major role in atherosclerosis development. Previously, we found that triglyceride (TG) promoted cell death of PMA-differentiated THP-1 macrophages. In this study, we compared the responsiveness of THP-1 monocytes and PMA-differentiated THP-1 macrophages to TNF-α-induced cell death. We found that, whereas THP-1 monocytes were TNF-α-resistant, THP-1 macrophages were sensitive to TNF-α-induced cell death. THP-1 monocytes treated with TG underwent cell death beginning at 24 h and addition of TNF-α further increased cell death. Based on these observations, we hypothesized that TG-induced differentiation of THP-1 monocytes into THP-1 macrophages, subsequently allowing sensitivity to TNF-α. To determine if TG could induce differentiation of THP-1 monocytes into THP-1 macrophages, we examined the mRNA expression levels of the macrophage-specific markers, CD11b, CD18, CD36 and CD68, by RT-PCR analysis. Our results show that expression of CD11b, CD36 and CD68 increased in TG-treated THP-1 monocytes in a dose- and time-dependent manner; furthermore, TNF-α expression was upregulated in TG-treated THP-1 monocytes. We have concluded that TG induces differentiation of THP-1 monocytes into macrophages concomitant with the production of TNF-α and increased sensitivity to TNF-α-dependent cell death.     

Transforming growth factor-beta 1 inhibits scavenger receptor activity in THP-1 human macrophages.

The macrophage scavenger receptor, a 220-kDa trimeric membrane glycoprotein, mediates the internalization of modified forms of low density lipoprotein (LDL) such as acetyl-LDL and oxidized-LDL and thus is likely to play a key role in atheroma macrophage foam cell formation. In addition, recent evidence suggests that the scavenger receptor may be an important macrophage binding site for lipopolysaccharide involved in lipopolysaccharide scavenging by macrophages. However, little is known about the regulation of this important receptor. We now report that the induction of scavenger receptor activity (as measured by acetyl-LDL stimulation of intracellular cholesterol esterification) seen in phorbol ester-differentiated THP-1 human macrophages was completely suppressed to the level seen in undifferentiated THP-1 monocytes by picomolar concentrations of transforming growth factor-beta 1 (TGF-beta 1). 125I-Acetyl-LDL degradation was inhibited in a dose-dependent manner by TGF-beta 1, with maximal inhibition (approximately 70%) occurring at 24 pM TGF-beta 1. Scatchard analysis revealed that TGF-beta 1 treatment resulted in a approximately 2-fold decrease in receptor number, and Northern blot analysis of RNA isolated from differentiated THP-1 macrophages demonstrated approximately 2-fold less scavenger receptor mRNA in TGF-beta 1-treated cells compared with that in macrophages not treated with TGF-beta 1. Since TGF-beta 1 is thought to be present in both atherosclerotic and inflammatory lesions, the above findings may have physiological relevance regarding the regulation of atheroma foam cell formation and/or the regulation of lipopolysaccharide clearance by macrophages.     

Galectin-3 expression in macrophages is signaled by Ras/MAP kinase pathway and up-regulated by modified lipoproteins

To study the signaling pathway involved in the regulation of galectin-3 expression we used phorbol ester to stimulate macrophage differentiation of THP-1 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) increased significantly the level of expression of galectin-3 in THP-1 cells. PMA-induced galectin-3 overexpression was blocked by: protein kinase C inhibitors staurosporine, calphostin C, and apigenin; tyrosine-specific protein kinase inhibitors genistein and tyrphostin A25; PD 98059, a selective inhibitor of mitogen-activated protein kinase (MAPK) kinase 1 (MEK1 or MKK1); and SB 203580, a specific inhibitor of p38 MAPK. Galectin-3 up-regulation was not affected by exposure to two inhibitors of cAMP-dependent protein kinase (PKA), H-89 and KT5720. Co-transfection of pPG3.5, a plasmid vector containing the rabbit galectin-3 promoter and the constructs pMCL-MKK1 N3 or pRC-RSV-MKK3Glu that constitutively express MKK1 and MKK3, raised the activity of galectin-3 promoter by 185% and 110%, respectively. Co-transfection with a Ha-Ras expression vector stimulated galectin-3 promoter activity approximately 10-fold. Expression of c-Jun or v-Jun raised the level of galectin-3 promoter activity more the three- and fourfold, respectively. Co-transfection of c-Jun and pPG3.5 5'-upstream deletion mutants resulted in a reduction of the galectin-3 promoter activity by 50% to 80%. Transfection of c-Jun, v-Jun or Ha-Ras increased significantly galectin-3 protein in THP-1 cells. These findings indicated that Ras/MEKK1/MKK1-dependent/AP-1 signal transduction pathway plays an important role in the expression of galectin-3 in PMA-stimulated macrophages. We further investigated the effect of modified lipoproteins on galectin-3 expression in macrophages. Murine resident peritoneal macrophages loaded with acetylated low-density lipoprotein (AcLDL) or oxidized LDL (OxLDL) showed increased galectin-3 protein and mRNA. These results showed that treatment of macrophages with PMA or modified lipoproteins results in galectin-3 overexpression. These findings may explain the enhanced expression of galectin-3 in atherosclerotic foam cells and suggest that Ras/MAPK signal transduction pathway is involved in controlling this gene.