A 63 kDa tumor necrosis factor inhibitor released from a human monocytic leukemia cell line, THP-1

A human monocytic cell line, THP-1-S, was cultured in a serum-free medium. The effect of the culture supernatant of THP-1-S on the cytotoxicity of rTNF-alpha to three kinds of cell lines and the binding of rTNF to its receptor were tested. The supernatant inhibited the cytotoxicity of rTNF-alpha when tested by the neutral red uptake method. In addition, the supernatant blocked the binding of 125I-rTNF-alpha to its receptor. Furthermore, following precipitation with PEG we detected complexes between rTNF-alpha and the inhibitory factor which formed during incubation with the culture supernatant from THP-1-S cells. However, the supernatant did not bind to or down-regulate the receptor for TNF-alpha on the cell surface of L-M-2d6 cells. This factor eluted with an apparent molecular mass of 63,000 Da by gel filtration and did not react with antibodies against p55 and p75 TNF receptors. These data suggest that human monocytic cells are capable of releasing an inhibitory factor against rTNF-alpha in serum-free culture conditions.     

Synergistic increases in IL-1 synthesis by the human monocytic cell line THP-1 treated with PAF and endotoxin

The capacity to stimulate cytokine release may be important to the long-term effects of platelet-activating factor (PAF), which has a very short half-life. Previous studies have shown that PAF stimulates interleukin 1 (IL-1) release by human monocytes. IL-1 and other cytokines produced in response to PAF may be important to the long-term effects of this short-lived lipid. The THP-1 human monocytic leukemia cell line, was used to study the mechanism by which PAF stimulates IL-1 release. PAF stimulates the release of IL-1 beta activity into THP-1 cell supernatants with a multiphasic dose-response curve very similar to that for monocytes. When THP-1 cells are treated with PAF and LPS in combination, these two stimuli interact synergistically to greatly increase the release of IL-1 activity. To assess the effect of PAF on IL-1 beta synthesis, THP-1 cell pellet proteins were separated by SDS-PAGE, blotted, and immunostained to detect IL-1 beta. Immunostaining revealed that PAF increases intracellular IL-1 beta precursor and that the combination of PAF and LPS increases IL-1 beta precursor synergistically. PAF increases IL-1 beta release mainly by increasing IL-1 beta synthesis.     

Induction of haptoglobin by all-trans retinoic acid in THP-1 human monocytic cell line

Haptoglobin (Hp) is one of the acute-phase proteins and is mainly synthesized in the liver. During our study on the differentiation of leukemia cells, we have found that Hp is synthesized in human monocytic cells by all-trans retinoic acid (ATRA). The synthesis of Hp by ATRA is induced in a dose- and time-dependent manner. Hp cDNA cloned from ATRA-treated THP-1 cells corresponds to the Hp alpha 2(FS)-beta form. Whereas ATRA acted as a strong inducer in THP-1 cells, IL-1 beta, TNF-alpha, IL-6, and LPS had little effect on Hp gene expression in these cells. These findings suggest that THP-1 cells express the Hp gene through a signal pathway different from hepatocytes, and that ATRA is a potent Hp-inducer in these cells.     

Thrombospondin-1 differentially regulates release of IL-6 and IL-10 by human monocytic cell line U937

We investigated possible regulatory effects of thrombospondin-1 (TSP-1), a multifunctional extracellular matrix protein, on cytokine release from macrophages. Immobilized TSP-1 enhanced IL-6 release from the human monocytic U937 cells stimulated with phorbol myristate acetate and LPS, whereas it inhibited IL-10 release. The 70-kDa fragment of TSP-1 containing the type 1 repeats showed the same regulatory effects. The enhanced IL-6 release by TSP-1 was inhibited by anti-CD36 antibody or antibody against the sequence of the binding site to CD36 in the type 1 repeats of TSP-1. Conversely, the decrease in IL-10 release by TSP-1 was strengthened by the blocking of the interaction between CD36 and TSP-1. Furthermore, the involvement of TGF-beta1 in the inhibition of IL-10 release by TSP-1 was indicated by the facts that (i) TSP-1 induced activation of TGF-beta1 produced by the U937 cells, (ii) exogenously added TGF-beta1 inhibited IL-10 release, and (iii) antibody against TGF-beta1 blocked the inhibition of IL-10 release by TSP-1. Together, the present findings suggest that TSP-1 enhances IL-6 release from macrophages by interaction with CD36, whereas IL-10 release is regulated by the balance between the enhancing effect of TSP-1 via CD36 and the suppressive effect by TSP-1-activated TGF-beta1.     

Scavenger receptor of human monocytic leukemia cell line (THP-1) and murine macrophages for nonenzymatically glycosylated proteins

Long-term incubation of proteins with glucose undergo a series of nonenzymatic reactions to form advanced glycosylation end product (AGE) with fluorescence and brown color. The receptor for AGE-proteins was demonstrated in murine macrophages (Vlassara et al. (1985) Proc. Natl. Acad. Sci. USA 82. 5588). Our recent study with rat macrophages revealed that the receptor also recognized proteins modified with aliphatic aldehydes such as formaldehyde or glycolaldehyde, indicating its close identity to a scavenger receptor for aldehyde-modified proteins (Takata, K. et al. (1988) J. Biol. Chem. 263. 14819). This notion was tested in the present study with human monocytic leukemia cell line (THP-1 cells), human monocyte macrophages and murine peritoneal macrophages. Endocytic uptake of AGE-proteins and aldehyde-modified proteins was inhibited in a cross-competitive fashion. The receptor activities of THP-1 cells for AGE-albumin and aldehyde-modified proteins were induced synchronously by phorbol 12-myristate 13-acetate. Furthermore, upon reduction by NaBH4 of the Schiff base formed between proteins and glucose or aldehydes, no ligand activity was generated. However, once the ligand activity was generated, NaBH4 was no longer effective for the ligand activity. Thus, a structure in common between AGE-proteins and aldehyde-modified proteins may be crucial for recognition by the human macrophage receptor.     

Independent regulation of IFN-gamma and tumor necrosis factor by IL-1 in human T helper cells

The lymphokines IL-2 and IL-4 promoted the growth of human PHA-triggered T cells, but only IL-2 induced the production of IFN-gamma and TNF. The addition of purified monocytes strongly enhanced the production of IFN-gamma in IL-2-stimulated T cell cultures but did not influence the production of TNF or the level of T cell proliferation. The addition of IL-1 to T cells activated by PHA and optimal concentrations of IL-2 resulted in a strong induction of IFN-gamma production but had no influence on TNF production or T cell proliferation. IL-6 did not influence IFN-gamma or TNF production or T cell proliferation induced by PHA-IL-2 and did not modulate IL-1-induced IFN-gamma production. The production of IFN-gamma by CD4+ 45R+ Th cells was strongly enhanced by IL-1, whereas CD8+ T cells were less responsive to IL-1 and CD4+ 45R+ T cells were unresponsive to IL-1. We demonstrate, at the clonal level, that the optimal production of IFN-gamma by human Th cells requires both IL-1 and IL-2, whereas the production of TNF and T cell proliferation are induced by IL-2 alone. We suggest that IL-1 acts as a second signal for IFN-gamma production and that it may have an important function in regulating the pattern of lymphokines produced by T cell subsets during activation.     

Detection of measles virus-induced apoptosis of human monocytic cell line (THP-1) by DNA fragmentation ELISA

Abstract Measles virus (wild strain, Toyoshima strain)-induced cell death is characterized by cell shrinkage, chromatin condensation, and nuclear fragmentation in a human monocytic cell line (THP-1). DNA fragmentation of measles virus-infected THP-1 cells was demonstrated by DNA agarose gel electrophoresis as well as by DNA fragmentation ELISA. When measles virus-infected THP-1 cells were cultured on monolayers of fibroblasts or human umbilical vein endothelial cells (HUVEC), the percentage of measles virus antigen-positive THP-1 cells and DNA fragmentation were significantly decreased. Addition of anti-intercellular adhesion molecule (ICAM)-1 (CD54) monoclonal antibody to culture of measles virus-infected THP-1 cells reduced significantly DNA fragmentation induced by measles virus. These findings suggest that inhibition of virus spread by fibroblasts and HUVEC reduces apoptosis, and ICAM-1 (CD54) may participate in the DNA fragmentation pathway.     

IL-4 regulates endothelial cell activation by IL-1, tumor necrosis factor, or IFN-gamma.

Alteration in the surface membrane of endothelial cells (EC) is a feature of endothelial activation both at sites of inflammation in vivo and after stimulation with cytokines in vitro. The effects of stimulating EC with IL-1 or TNF include enhanced adhesiveness for polymorphonuclear leukocytes (PMN) and T cells, the induction of EC leukocyte adhesion molecule-1 (ELAM-1) expression, and the increased expression of intercellular adhesion molecule-1 (ICAM-1) and the 1.4C3 Ag. In contrast, IFN-gamma stimulation increases EC binding of T cells but not PMN and enhances ICAM-1 expression but not ELAM-1 or 1.4C3 Ag expression. Recently we have reported that the T cell-derived cytokine IL-4 also increases EC adhesiveness for T cells but not PMN. In this study we have examined the effect of IL-4 on the expression of several cytokine-inducible EC activation Ag, by using a previously described ELISA technique. IL-4 modulation of activation Ag expression was concentration dependent, optimal at around 100 U/ml, and exhibited a unique pattern compared to that seen with the other cytokines. Although, IL-4 stimulation increased 1.4C3 Ag expression (p less than 0.001), it significantly inhibited constitutive ICAM-1 expression (p less than 0.01) and did not induce ELAM-1. Furthermore, IL-4 exhibited significant synergy with IL-1 or TNF in inducing 1.4C3 Ag expression (p less than 0.001) but inhibited the increased expression of ICAM-1 produced by IL-1, TNF, or IFN-gamma (p less than 0.01) and inhibited the induction of ELAM-1 by IL-1 and TNF (p less than 0.001). In contrast, IL-4 had no effect on the expression of EC HLA-class I, -DR, -DP, or -DQ and neither enhanced nor inhibited the effect of IFN-gamma on the expression of these molecules. Finally, although IL-4 alone caused little if any shape change in EC monolayers, it strongly synergized with TNF or IFN-gamma in causing a change in shape to a more fibroblastic morphology. These observations indicate that IL-4 increases EC adhesiveness for T cells by the induction of a different adhesion molecule to ICAM-1. Furthermore, the ability of IL-4 to both enhance and inhibit the expression of activation Ag on EC already activated by IL-1, TNF, or IFN-gamma suggests that it may be important in altering the quality of inflammatory responses such as may occur during the development and maintenance of chronic or immune-mediated inflammation.     

Differential regulation of the human IFN-gamma receptor expression in Raji and IM9 lymphoblastoid cells versus THP-1 monocytic cells by IFN-gamma and phorbol myristate acetate

Expression of the human (hu) IFN-gamma-R has been studied in Raji and IM9 cells (two B lymphoblastoid cell lines) and in THP-1 cells (a monocytic cell line) with respect to IFN-gamma binding sites, receptor protein and mRNA levels. Although, in these three cell lines, the hu-IFN-gamma-R mRNA was expressed to the same extent, the high affinity receptor was expressed differently both in cell surface receptor binding and amount of receptor protein. Various ligands are able to modulate the expression of their own receptor. We investigated the modulation of the hu-IFN-gamma-R by its ligand. Hu-IFN-gamma induced a rapid and dose-dependent decrease of its cell surface receptor number without alteration of receptor affinity, amounts of receptor protein or hu-IFN-gamma-R mRNA accumulation and stability. Thus, in Raji, IM9, and THP-1 cells, the hu-IFN-gamma had no effect on its receptor gene expression and the cell surface decrease was simply due to ligand blocking and receptor internalization rather than true down-regulation. The second messenger in the hu-IFN-gamma signal transduction pathway is not well characterized, but activation of protein kinase C has been reported in some cases. Therefore, the modulation of the hu-IFN-gamma-R expression by PMA, a potent activator of protein kinase C and a modulator of other receptor expression, has been investigated. In Raji and IM9 cells, PMA had no or few effects on the cell surface receptor number and no detectable effect on the receptor protein or on mRNA levels. In contrast, in THP-1 cells, PMA treatment induced a time and dose-dependent five- to sixfold increase of the cell surface receptors due to a rapid and persistent increase of the hu-IFN-gamma-R gene expression in THP-1 cells was specifically inhibited or reversed by hu-IFN-gamma treatment. The modulation of the hu-IFN-gamma-R expression by PMA in THP-1 cells and by hu-IFN-gamma in PMA-treated THP-1 cells seems associated with their effect on monocyte-macrophage differentiation and/or macrophage activation.     

Coxsackievirus B3-induced production of tumor necrosis factor-alpha, IL-1 beta, and IL-6 in human monocytes.

Infections by coxsackievirus B3 (CVB3) have previously been shown to cause acute and chronic myocarditis characterized by a heavy mononuclear leukocyte infiltration and myocyte necrosis. Because clinical and experimental evidence suggested that cardiac damage may result from immunologic rather than viral mechanisms, we examined in this study the in vitro interaction of CVB3 with human monocytes. CVB3 was capable of infecting freshly harvested monocytes as revealed by immunofluorescence and release of infectious virus particles. Virus infection did not reduce monocyte viability but, on the contrary, enhanced spreading and adherence. In a dose-dependent manner, CVB3 stimulated the release of cytokines from monocytes. Whereas a potent production of TNF-alpha, IL-1 beta, and IL-6 was dependent on exposure to infectious CVB3, IFN release was also induced by UV-inactivated virus. On a molecular level, CVB3 stimulated cytokine gene expression as shown by a marked TNF-alpha, IL-1 beta, and IL-6 mRNA accumulation. Supernatants of CVB3-infected monocytes displayed cytotoxic activity against Girardi heart cells which could be abrogated by an anti-TNF-alpha antiserum. These data suggest that CVB3-induced cytokine release from monocytes may participate in virus-induced organ damage such as myocarditis, which may either occur by a direct cytotoxicity of cytokines or by activation of cytotoxic lymphocytes.     

Methotrexate induces production of IL-1 and IL-6 in the monocytic cell line U937

Introduction

Methotrexate (MTX) has been for decades a standard treatment in a wide range of conditions, from malignancies to rheumatoid arthritis (RA). Despite this long experience, the mechanisms of action of MTX remain incompletely understood. Reported immunologic effects of MTX include induction of increased production of some cytokines, an effect that seems to be at odds with the generally anti-inflammatory effects of this drug in diseases like RA. To further elucidate these immune activities, we examined effects of MTX on the human monocytic cell line U937.

Methods

The U937 cell line was treated in vitro with pharmacologic-range concentrations of MTX and effects on production of interleukin (IL)-1, IL-6 and TNF alpha were measured. Changes in gene expression for IL-1 and IL-6 and specificities in the Jun-N-terminal kinase (JNK) signaling pathway including JNK 1, JNK2, JUN and FOS were also determined. The contribution of NF-kB, folate and adenosine pathways to the observed effects was determined by adding appropriate inhibitors to the MTX cultures.

Results

MTX mediated a dose-dependent increase in IL-1 and IL-6 in U937 cells, as measured by secreted proteins and levels of gene expression. The increased cytokine expression was inhibited by addition of parthenolide and folinic acid, but not by caffeine and theophylline, suggesting that NF-kB and folates, but not adenosine, were involved in mediating the observed effects. When U937 cells were cultured with MTX, upregulated expression of JUN and FOS, but not JNK 1 or 2, also was observed.

Conclusions

MTX induces expression of proinflammatory cytokines in U937 monocytic cells. These effects might mediate the known toxicities of MTX including pneumonitis, mucositis and decreased bone mineral density.     

Contribution of IL-6 to the antiproliferative effect of IL-1 and tumor necrosis factor on tumor cell lines

The role of IL-6 in the antiproliferative effect of IL-1 for tumor cell lines was investigated using IL-1-sensitive cell lines. Human recombinant IL-1 alpha and IL-6 both inhibited the growth of an IL-1-sensitive cloned human melanoma cell line (A375-C6). However, IL-1 has greater maximum growth inhibitory activity than IL-6. Conditioned medium of the tumor cells that were treated with IL-1 contained IL-6 as determined by ELISA. Northern blot analysis revealed that IL-6 mRNA expression increased in IL-1-treated cells. In addition, antibody against human IL-6 neutralized about 50% of the antiproliferative effect of IL-1. The growth of an IL-1-resistant clone of A375 cells (A375-C5), which cannot be shown to express any detectable IL-1R, was inhibited by IL-6 to the same degree as A375-C6 cells. The A375-C5 cell line did not produce IL-6 or increase IL-6 mRNA after stimulation with IL-1. These results indicate that IL-6 mediates in part the antiproliferative effect of IL-1 on A375-C6 cells by acting as an autocrine antiproliferative factor. IL-1 also inhibited the growth of a malignant human mammary cell line (MDA-MB-415). IL-6 exhibited only slight growth inhibition in this cell line. Neither IL-6 production nor IL-6 mRNA expression was induced in this cell line by IL-1. Antibody against IL-6 did not neutralize the antiproliferative effect of IL-1. Therefore, for MDA-MB-415 cells IL-6 appeared not to be involved in the antiproliferative effect of IL-1. These results indicate that the antiproliferative effect of IL-1 involves at least two pathways, one IL-6 dependent and another IL-6 independent. The contribution of IL-6 to the antiproliferative effect of TNF was also examined. IL-6 appeared not to play a role in the antiproliferative effect of TNF in these cell lines.     

Isolation and identification of C-type natriuretic peptide in human monocytic cell line, THP-1.

In a survey for unknown bioactive peptides in mammalian cell lines, we isolated peptides exhibiting a strong relaxant effect on chick rectum from a phorbol ester-supplemented culture medium of the human monocytic cell line, THP-1. The peptide was deduced to be a C-terminal 29-residue peptide derived from a human C-type natriuretic peptide (CNP) precursor. CNP mRNA was also detected in the THP-1 cells, and expression of CNP gene and CNP concentration in the culture medium was found to be highly augmented by the stimulation of phorbol ester. Production of CNP in THP-1 cells suggests that CNP also functions as a local regulator in the blood cell-vascular system, although CNP has previously been recognized as a neuropeptide functioning in the central nervous system.     

IL-6 and tumor necrosis factor-alpha. Autocrine and paracrine cytokines involved in B cell function

IL-6 and TNF-alpha are synthesized and secreted by normal tonsillar B cells after stimulation with the polyclonal B cell activator Staphylococcus aureus Cowan strain 1 (SAC) and IL-2 as well as spontaneously by in vivo activated B cells from patients with hypergammaglobulinemia. Using specific neutralizing antibodies, both factors were shown to be involved in autocrine and/or paracrine regulation of B cell differentiation. IgG induced by SAC/IL-2 stimulation was reduced 73% with an anti-IL-6 antibody and 40% with an anti-TNF-alpha antibody. Similar effects of these antibodies were observed on the spontaneous in vitro IgG production by lymphoblastic B cells from six patients with hypergammaglobulinemia. Kinetic studies with SAC/IL-2-activated B cells revealed that the anti-TNF-alpha antibody must be present at the beginning of the culture to exert an effect on Ig production, whereas the anti-IL-6 antibody reduced Ig production even if added as late as day 3. This sequential action of TNF-alpha and IL-6 on B cell differentiation was reflected by different kinetics of release of these two cytokines into the supernatant of SAC/IL-2 activated B cells; TNF-alpha peaked at 24 h and IL-6 at 96 h after stimulation. In addition, it was shown that IL-6 production by in vitro-activated B cells was partially blocked by an anti-TNF-alpha antibody suggesting that TNF-alpha regulates IL-6 production in normal B cells via an autocrine pathway. We also investigated the effects of TGF-beta on TNF-alpha and IL-6 production by normal B cells. Although TGF-beta inhibited Ig production by in vitro-activated and in vivo-activated B cells, it did not inhibit the release of these cytokines from normal B cells. Furthermore, TGF-beta did not inhibit the induction of nuclear factor-IL-6 nor the expression of IL-6R on activated B cells. Thus, although the biologic effects of anti-IL-6 and TGF-beta on B cell Ig production are similar, their mechanisms of actions appear to be distinct.