Variables that regulate production of insulin-like peptide(s) in human leukemia cell line (HL-60)

A human myeloid leukemia cell line (HL-60) produces a peptide or peptides with insulin-like activity which is distinct from insulin or insulin-like growth factors (somatomedins). Factors regulating the production of this peptide (HL-ILP) were explored in the present study. The production of HL-ILP was maximal in the early log phase of cell growth and declined with increasing cell density. Differentiation of HL-60 cells to macrophages, induced by dihydroxyvitamin D3 or phorbol esters, was also associated with a decrease in HL-ILP production. Glucose consumption by the cells in the early log phase was closely related with HL-ILP production, and HL-ILP was found to stimulate glucose consumption by HL-60 cells. Production of HL-ILP was dependent on glucose concentrations in the culture medium and glucose concentrations higher than 1mg/d1 suppressed the release of HL-ILP. These observations are not inconsistent with a hypothesis that HL-ILP is involved in the glucose metabolism of the HL-60 cells that produce this peptide.     

THP-1 cells form foam cells in response to coculture with lipoproteins but not platelets

The human monocytic leukemia cell line, THP-1, shares many properties with human monocyte-derived macrophages and might be a useful model for studying foam cell formation in vitro. Therefore, we examined the ability of THP-1 cells to accumulate cholesteryl esters, the hallmark feature of foam cells, in response to culture with native low density lipoprotein (LDL), modified LDL, and platelets. THP-1 cells stored more cholesteryl esters than macrophages in response to 200 micrograms/ml of LDL. Down-regulation of LDL receptors occurred in macrophages at lower LDL concentrations than in THP-1 cells. Phorbol ester-treated THP-1 cells stored more cholesteryl esters than human macrophages in response to 25-200 micrograms/ml of acetylated LDL. Because we have previously demonstrated that activated platelets enhanced macrophage cholesteryl ester storage, we examined the ability of THP-1 cells to store cholesteryl esters in response to coculture with platelets. Compared with macrophages, dividing THP-1 cells and phorbol ester-treated THP-1 cells accumulated only 50% and 33% as much cholesteryl esters, respectively. Furthermore, although platelets induced a 90% reduction in cholesterol synthesis in macrophages by day 5, cholesterol synthesis in THP-1 cells and phorbol ester-treated THP-1 cells was inhibited less than 50% by platelets. Nevertheless, both THP-1 cells and macrophages responded to platelets by increasing their secretion of apolipoprotein E. Therefore, we conclude that dividing THP-1 cells and phorbol ester-treated THP-1 cells are capable of forming foam cells in response to physiologic doses of both LDL and acetylated LDL, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activity of tissue factor in microparticles produced <Emphasis Type="Italic">in vitro</Emphasis> by endothelial cells,monocytes, granulocytes,and platelets

Activity of tissue factor (TF) in membrane microparticles (MPs) produced in vitro by endothelial cells (ECs), monocytes, THP-1 monocytic cells, granulocytes, and platelets was investigated. ECs were isolated from human umbilical vein, and monocytes, granulocytes, and platelets–from the blood of healthy donors. ECs, monocytes, and THP-1 cells were activated by bacterial lipopolysaccharide, granulocytes–by lipopolysaccharide or phorbol myristate acetate, and platelets - by SFLLRN, thrombin receptor-activating peptide. MPs were sedimented from the culture medium or supernatant of activated cells at 20,000g for 30 min. Coagulation activity of MPs was analyzed in a modified recalcification assay by assessing their effects on coagulation of donor plasma depleted of endogenous MPs (by centrifuging at 20,000g for 90 min). MPs from all cell types accelerated plasma coagulation. Antibodies blocking TF activity prolonged coagulation lagphase in the presence of MPs from ECs, monocytes, and THP-1 cells (by 2.7-, 2.0-, and 1.8-fold, respectively), but did not influence coagulation in the presence of MPs from granulocytes and platelets. In accordance with these data, TF activity measured by its ability to activate factor X was found in MPs from ECs, monocytes, and THP-1 cells, but not in MPs from granulocytes and platelets. The data obtained indicate that active TF is present in MPs produced in vitro by ECs, monocytes, and THP-1 cells, but not in MPs derived from granulocytes and platelets.     

C-type natriuretic peptide receptor expression in pancreatic alpha cells

Atrial natriuretic peptide (ANP), brain type natriuretic peptide (BNP) and C-type natriuretic peptide (CNP) comprise a family of natriuretic peptides that mediate their biological effects through three natriuretic peptide receptor subtypes, NPR-A (ANP, BNP), NPR-B (CNP) and NPR-C (ANP, BNP, CNP). Several reports have provided evidence for the expression of ANP and specific binding sites for ANP in the pancreas. The purpose of this study was to identify the ANP receptor subtype and to localize its expression to a specific cell type in the human pancreas. NPR-C immunoreactivity, but neither ANP nor NPR-A, was detected in human islets by immunofluorescent staining. No immunostaining was observed in the exocrine pancreas or ductal structures. Double-staining revealed that NPR-C was expressed mainly in the glucagon-containing alpha cells. NPR-C mRNA and protein were detected in isolated human islets by RT-PCR and Western blot analysis, respectively. NPR-C expression was also detected by immunofluorescent staining in glucagonoma but not in insulinoma. ANP, as well as BNP and CNP, stimulated glucagon secretion from perifused human islets (1,111 ± 55% vs. basal [7.3 fmol/min]; P < 0.001). This response was mimicked by cANP(4–23), a selective agonist of NPR-C. In conclusion, the NPR-C receptor is expressed in normal and neoplastic human alpha cells. These findings suggest a role for natriuretic peptides in the regulation of glucagon secretion from human alpha cells.     

脓毒症单核巨噬细胞特异性结合肽的筛选

利用噬菌体随机肽库展示技术,筛选出与脓毒症单核/巨噬细胞特异性结合的短肽,探索脓毒症治疗的新方法.分别以经过脂多糖(lipopolysaccharide, LPS)处理的人外周血单核细胞株(THP-1)细胞作为筛选的靶细胞,以未经LPS处理的THP-1细胞作为非特异性噬菌体吸附细胞,对噬菌体随机环七肽库进行4轮“差减"筛选,经过细胞ELISA验证阳性噬菌体克隆,对获得的阳性克隆进行DNA测序及生物信息学分析,并进一步利用免疫荧光实验,鉴定噬菌体克隆与LPS处理THP-1细胞的结合特异性.4轮筛选后,随机挑取的噬菌体克隆,测序后得到可与LPS处理的THP-1细胞特异性结合肽.对去冗余后的七肽进行Clustal W多序列比对分析和BlastP蛋白同源相似性分析,细胞免疫荧光检测确定获得的噬菌体展示七肽可与LPS处理的THP-1细胞特异性结合.噬菌体随机肽库技术为脓毒症单核/巨噬细胞表面靶位的筛选提供了高效、快捷的筛选体系,实验获得的多肽基序具有高度保守性和细胞特异性,这些多肽的生物活性将是下一步的研究内容.     

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.     

Natriuretic peptides reduce plasminogen activator inhibitor-1 expression in human endothelial cells

Plasma concentrations of natriuretic peptides increase in some pathological conditions, but very little is known about the effect of these vasodilator peptides on the regulation of the blood coagulation system. The fundamental role in the regulation of fibrinolysis is played by plasminogen activator inhibitor type 1 (PAI-1). Recent studies demonstrate that natriuretic peptides can modulate PAI-1 expression in bovine aortic smooth muscle cells and rat aortic endothelial cells. In this report, we tested the effect of natriuretic peptides on PAI-1 expression in the human endothelial cell line (EA.hy 926). For this purpose, we treated the cell cultures with ANP, BNP and CNP, and modulation of PAI-1 synthesis was evaluated. We compared the effect of natriuretic peptides on synthesis and release of PAI-1 in unstimulated cells, and after activation with tumour necrosis factor alpha (TNFalpha). Natriuretic peptides abolished TNFalpha - induced upregulation of PAI-1 expression at both the PAI-1 mRNA and the antigen levels. The inhibitory efficiency was higher in the case of CNP when compared to that produced by ANP and BNP, particularly when TNFalpha-stimulated cells were used. We observed an inhibition of stimulatory effect of TNFalpha on PAI-1 expression also at the level of the PAI-1 promoter in cells transfected with a PAI-1 promoter fragment (+71 to -800). The PAI-1 promoter activity was markedly inhibited by C-type natriuretic peptide, already at a very low (0.001 micro M) concentration of the peptide.     

Establishment of an In vitro System to Study Intracellular Behavior of Candida glabrata in Human THP-1 Macrophages

A cell culture model system, if a close mimic of host environmental conditions, can serve as an inexpensive, reproducible and easily manipulatable alternative to animal model systems for the study of a specific step of microbial pathogen infection. A human monocytic cell line THP-1 which, upon phorbol ester treatment, is differentiated into macrophages, has previously been used to study virulence strategies of many intracellular pathogens including Mycobacterium tuberculosis. Here, we discuss a protocol to enact an in vitro cell culture model system using THP-1 macrophages to delineate the interaction of an opportunistic human yeast pathogen Candida glabrata with host phagocytic cells. This model system is simple, fast, amenable to high-throughput mutant screens, and requires no sophisticated equipment. A typical THP-1 macrophage infection experiment takes approximately 24 hr with an additional 24-48 hr to allow recovered intracellular yeast to grow on rich medium for colony forming unit-based viability analysis. Like other in vitro model systems, a possible limitation of this approach is difficulty in extrapolating the results obtained to a highly complex immune cell circuitry existing in the human host. However, despite this, the current protocol is very useful to elucidate the strategies that a fungal pathogen may employ to evade/counteract antimicrobial response and survive, adapt, and proliferate in the nutrient-poor environment of host immune cells.     

Rapid turnover of low density lipoprotein receptor in human monocytic THP-1 cells.

We examined whether human monocyte-derived macrophages had low density lipoprotein (LDL) receptors with a short life span. The human monocytic leukemia cell line, THP-1, was highly differentiated when treated with phorbol ester. LDL receptors degraded rapidly with half-lives of 3-4 h in THP-1 cells before phorbol ester treatment. During the transition into monocytic cells, expression of the LDL receptor gene was not affected. However, relative degradation rates of LDL receptors normalized by those of cellular total proteins were about twice as fast in phorbol ester-treated THP-1 cells compared to untreated cells.     

Dendroaspis natriuretic peptide and its functions in pig ovarian granulosa cells

Dendroaspis natriuretic peptide (DNP), a 38-amino acid peptide, was isolated from the venom of green mamba. It has structural and functional similarities to the other members of the natriuretic peptide family. The purpose of this study was to determine whether DNP is present in pig ovarian granulosa cells and to define its biological functions. The serial dilution curves of extracts of granulosa cells and follicular fluid were parallel to the standard curve of DNP, and a major peak of molecular profile of both extracts by HPLC was synthetic DNP. The concentration of DNP was 7.51+/-1.46 pg/10(7) cells and 24.81+/-2.38 pg/ml in granulosa cells and follicular fluid, respectively. Natriuretic peptides increased cGMP production in the purified membrane of granulosa cells with a rank order of potency of C-type natriuretic peptide (CNP)>atrial natriuretic peptide (ANP)=DNP. mRNAs for natriuretic peptide receptor-A (NPR-A), NPR-B and NPR-C were detected by RT-PCR. The binding site of (125)I-DNP was also observed in granulosa cell layer by in vitro autoradiography. Synthetic DNP inhibited the secretion of ANP from granulosa cells in a concentration-dependent manner and the potency was similar to CNP. The concentration of DNP and CNP, which inhibited the secretion of ANP by 50%, was about 1 nM. Increases in production of cGMP in granulosa cells were observed by DNP or CNP. Therefore, these results show the existence of DNP system and the cross-talk between natriuretic peptides in pig ovarian granulosa cells.     

A synthetic peptide mimic of plasma apolipoprotein E that binds the LDL receptor.

Human plasma apolipoprotein E (apoE) is a low density lipoprotein (LDL) receptor ligand. It targets cholesterol-rich lipoproteins to LDL receptors on both hepatic and peripheral cells. The region of apoE responsible for its binding to the LDL receptor has been localized to amino acids 140-160. An apoE 141-155 monomeric peptide and a dimeric 141-155 tandem peptide were synthesized and tested for their inhibition of 125I-LDL degradation by human fibroblasts and human monocytic-like cells, THP-1. The monomer had no activity at 250 microM, but the dimer inhibited 125I-LDL degradation by 50% at 5 microM. The inhibition was specific for the LDL receptor because the dimer did not inhibit the degradation of 125I-acetylated LDL by scavenger receptors expressed by phorbol ester-stimulated THP-1 cells. As reported for native apoE, amino acid substitutions of Lys-143----Ala, Leu-144----Pro, and Arg-150----Ala decreased the inhibitory effectiveness of the dimer. Furthermore, a trimer of the 141-155 sequence had a 20-fold greater inhibitory activity than the dimer. Studies with a radioiodinated dimer indicated that some of the inhibitory activity could be a result of the interaction of the dimer with LDL. However, direct binding of the 125I-dimeric peptide to THP-1 cells was observed as well. This binding was time-dependent, linear with increasing cell number, Ca(2+)- but not Mg(2+)-dependent, saturable, inhibited by lipoproteins, and increased by preculture of the cells in lipoprotein-depleted medium. Therefore, a synthetically prepared dimeric repeat of amino acid residues 141-155 of apoE binds the LDL receptor.     

Identification of a chemotactic epitope in human transforming growth factor-β1 spanning amino acid residues 368–374

TGF-b?1 plays a critical role in inflammatory and repair processes due in part to its ability to provide a potent chemotactic stimulus for inflammatory cells such as neutrophils and monocytes and for fibroblasts which initiate the fibrogenic response. In the present study, we have used synthetic oligopeptides representing the amino acid sequence of the 12.1 kDa monomer of human TGF-b?1 in an effort to identify a chemotactic epitope on the molecule. A seven residue peptide containing residues 368-374, Val Tyr Tyr Val Gly Arg Lys, was demonstrated to be capable of inducing chemotactic migration of human peripheral blood neutrophils, monocytes, monocyte leukemia cell line THP-1, and infant foreskin fibroblasts. Furthermore, larger peptides from the carboxy-terminal portion of TGF-b?1 that contained residues 368–374 also induced migration of these cell types. None of the peptides representing the complete amino acid of TGF-b?1 monomer were able to compete with [¹²⁵I]hrTGF-b?1 for binding to TGF-b? cell surface receptors or fibroblasts or THP-1 cells. Implications of these observations are discussed. © 1995 Wiley-Liss, Inc.     

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.     

C-型钠尿肽与血管损伤性疾病

C-型钠尿肽(C-type natriuretic peptide, CNP)作为钠尿肽家系的一员, 主要是由血管内皮分泌,CNP与血管平滑肌细胞钠尿肽受体-B(NPR-B)结合,激活颗粒型鸟苷酸环化酶,促进细胞内cGMP 水平升高,以旁分泌和/或自分泌方式调节循环系统功能稳态.CNP广泛分布于血管系统,尤其在内皮细胞中高表达.CNP具有利钠、利尿、调节血管张力、抑制血管平滑肌细胞迁移、增殖等作用,与高血压、动脉粥样硬化、血栓形成、冠脉成形术后再狭窄和血管钙化等多种血管损伤性疾病密切相关.     

C-type natriuretic peptide is a potent activator of guanylate cyclase in endothelial cells from brain microvessels.

An exposure of endothelial cells from rat brain microvessels to C-type natriuretic peptide (CNP) resulted in a rapid and large increase in cGMP formation. The action of CNP did not require inhibitors of phosphodiesterases to be observed and occurred at nanomolar concentrations. Other natriuretic peptides (ANP and BNP) also stimulated cGMP formation in endothelial cells from brain microvessels but with a potency that was at least 100 times less than that of CNP. In contrast, endothelial cells from the aorta showed large cGMP responses to low concentrations of ANP and BNP but were unresponsive to CNP up to concentrations as large as 100 nM. It is concluded that endothelial cells from brain microvessels and from aorta express different receptors subtypes for natriuretic peptides. Endothelial cells from brain microvessels express CNP specific ANPB receptors; aortic endothelial cells express ANP (and BNP) specific ANPA receptors. CNP may play an important role in the regulation of water and electrolyte movements across the blood brain barrier.     

C-type natriuretic peptide (CNP) effects on intracellular calcium [Ca2+]i in mouse gonadotrope-derived alphaT3-1 cell line.

C-type natriuretic peptide (CNP), the third member of the atrial natriuretic peptide family, acts via guanylyl cyclase containing GC-B receptors to stimulate cyclic guanosine 3',5' monophosphate (cGMP) accumulation in the gonadotrope-derived alphaT3-1 cell line and rat pituitary cells. This effect is inhibited by concomitant activation of the phospholipase C (PLC)-coupled gonadotrophin hormone-releasing hormone (GnRH) receptors in these cells. Since GnRH stimulates gonadotrophin secretion from gonadotropes by increasing the cytosolic Ca2+ concentration ([Ca2+]i) and natriuretic peptides have been found to influence PLC/Ca2+ signalling in other systems, we have investigated whether CNP can alter basal or GnRH-stimulated changes in [Ca2+]i in alphaT3-1 cells. In Ca 2+-containing medium, 10(-7) M CNP modestly, but significantly increased [Ca2+]i over several min, but subsequently inhibited the elevation of [Ca2+]i in response to 10(-7) M GnRH in both Ca2+-containing and Ca2+-free medium. This inhibitory effect was mimicked by 10(-6) M 8-Br-cGMP, but not by ANP, indicating mediation by cyclic GMP and the CNP-specific GC-B receptor. However, basal and GnRH-stimulated inositol (1,4,5) trisphosphate (Ins(1,4,5)P3) generation were not measurably affected by CNP, and CNP failed to affect thapsigargin-induced capacitative Ca2+ entry. Thus, it appears that the cross-talk between CNP and GnRH in these cells is reciprocal in that GnRH modulates CNP effects on cGMP generation, whereas, CNP modulates GnRH effects on Ca2+ mobilisation.     

Extracellular domain-IgG fusion proteins for three human natriuretic peptide receptors. Hormone pharmacology and application to solid phase screening of synthetic peptide antisera.

The natriuretic peptide receptors (NPRs) are a family of three cell surface glycoproteins, each with a single transmembrane domain. Two of these receptors, designated NPR-A and NPR-B, are membrane guanylyl cyclases that synthesize cGMP in response to hormone stimulation. The third receptor, NPR-C, has been reported to function in the metabolic clearance of ligand and in guanylyl cyclase-independent signal transduction. We engineered three chimeric proteins consisting of the natriuretic peptide receptor extracellular domains fused to the Fc portion of human IgG-gamma 1. These molecules provide material for detailed studies of the human receptor's extracellular domain structure and interaction with the three human natriuretic peptides, atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), and type-C natriuretic peptide (CNP). The homodimeric fusion proteins, designated A-IgG, B-IgG, and C-IgG, were secreted from Chinese hamster ovary cells and purified by protein-A affinity chromatography. We present here the primary characterization of these fusion proteins as represented by the intrinsic hormone affinities measured by saturation binding and competition assays. The dissociation constant of 125I-ANP for A-IgG was 1.6 pM and for C-IgG, 1.2 pM. The dissociation constant of 125I-Y0-CNP (CNP with addition of tyrosine at the amino terminus) for B-IgG was 23 pM. The rank order of potency in competitive binding for A-IgG was ANP greater than BNP much greater than CNP, whereas for B-IgG the ranking was CNP much greater than ANP greater than BNP. For C-IgG, we observed ANP greater than CNP greater than or equal to BNP. These data demonstrate that the receptor-IgG fusion proteins discriminate among the natriuretic peptides in the same manner as the native receptors and provide a basis for future structural studies with these molecules. The purified fusion proteins have a variety of potential applications, one of which we illustrate by a solid phase screening assay in which rabbit sera from a series of synthetic-peptide immunizations were titered for receptor reactivity and selectivity.     

Induction of acetyl-LDL receptor activity by phorbol ester in human monocyte cell line THP-1

Human monocytic cell line THP-1 incubated with as little as 10 ng/ml of phorbol myristate acetate bound and metabolized 1-2 micrograms of Ac-LDL over a 5-h period. In the absence of phorbol treatment, no specific metabolism of Ac-LDL occurred. Optimal levels of receptor were reached after 72 h of exposure. Induction of receptor was dependent on protein and RNA synthesis and was partially reversed upon removal of the phorbol. Induction of receptor required activation of the protein kinase C pathway. Metabolism of Ac-LDL by THP-1 cells at 37 degrees C was saturated at 25 micrograms/ml. Binding at 4 degrees C was saturable with an average Kd of 8.0 x 10(-9) M. Cell population studies by fluorescent activated cell sorting indicated that approximately 87% of the THP-1 population was expressing scavenger receptor activity 96 h after phorbol treatment as compared to 99% for murine macrophage cell line P388D1. Uptake of Ac-LDL by THP-1 resulted in an 11-fold increase in the rate of cholesterol esterification which was saturable at 50 micrograms/ml. Incubation of cells for 48 h with 50 micrograms/ml of Ac-LDL resulted in a 60% increase in free cholesterol and a 10-fold increase in the cholesteryl ester content of the cells. Lipid accumulation in THP-1 cells after Ac-LDL uptake was readily visible by Oil Red-O staining. Solubilization of THP-1 cells, before and after phorbol treatment, followed by ligand blotting with Ac-LDL detected the presence of a 250-kDa protein only in cells treated with phorbol. The protein comigrated with the scavenger receptor derived from mouse macrophage cell line P388D1.