Scoparone inhibits PMA-induced IL-8 and MCP-1 production through suppression of NF-kappaB activation in U937 cells

Scoparone is a major component of the shoot of Artemisia capillaris (Compositae), which has been used for the treatment of hepatitis and biliary tract infection in oriental countries. In this study, the effects of scoparone on the expression of interleukin-8 (IL-8) and monocyte chemotactic protein-1 (MCP-1) and activation of nuclear factor-kappaB (NF-kappaB) were examined in U937 human monocytes activated with phorbol 12-myristate 13-acetate (PMA). Scoparone (5-100 microM) had no cytotoxic effect in unstimulated cells and concentration-dependently reversed PMA-induced toxicity in the cells stimulated with PMA. Scoparone concentration-dependently reduced the release of IL-8 and MCP-1 protein and expression of IL-8 and MCP-1 mRNA levels induced by PMA. Moreover, scoparone inhibited the levels of NF-kappaB-DNA complex and NF-kappaB activity in the cells stimulated with PMA in a concentration-dependent manner. Scoparone dose-dependently inhibited the phosphorylation of IkappaBalpha and nuclear translocation of NF-kappaB1 p50, RelA p65, and c-Rel p75. These data suggest that scoparone may inhibit the expression of chemokines (IL-8 and MCP-1) in PMA-stimulated U937 cells and a potential mechanism of scoparone may be inhibition of NF-kappaB activation, which is linked to inhibition of NF-kappaB subunits (NF-kappaB1 p50, RelA p65, and c-Rel p75) translocation via suppression of IkappaBalpha phosphorylation.     

Involvement of p42/p44 MAPK, JNK, and NF-kappaB in IL-1beta-induced ICAM-1 expression in human pulmonary epithelial cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of intercellular adhesion molecule-1 (ICAM-1) on airway epithelial cells and contributes to inflammatory responses. However, the mechanisms regulating ICAM-1 expression by IL-1beta in human A549 cells was not completely understood. Here, the roles of mitogen-activated protein kinases (MAPKs) and NF-kappaB pathways for IL-1beta-induced ICAM-1 expression were investigated in A549 cells. IL-1beta induced expression of ICAM-1 protein and mRNA in a time- and concentration-dependent manner. The IL-1beta induction of ICAM-1 mRNA and protein were partially inhibited by U0126 and PD98059 (specific inhibitors of MEK1/2) and SP600125 [a specific inhibitor of c-Jun-N-terminal kinase (JNK)]. U0126 was more potent than other inhibitors to attenuate IL-1beta-induced ICAM-1 expression. Consistently, IL-1beta stimulated phosphorylation of p42/p44 MAPK and JNK which was attenuated by pretreatment with U0126 or SP600125, respectively. Moreover, transfection with dominant negative mutants of MEK1/2 (MEK K97R) or ERK2 (ERK2 K52R) also attenuated IL-1beta-induced ICAM-1 expression. The combination of PD98059 and SP600125 displayed an additive effect on IL-1beta-induced ICAM-1 gene expression. IL-1beta-induced ICAM-1 expression was almost completely blocked by a specific NF-kappaB inhibitor helenalin. Consistently, IL-1beta stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha which was blocked by helenalin, U0126, or SP600125. Taken together, these results suggest that activation of p42/p44 MAPK and JNK cascades, at least in part, mediated through NF-kappaB pathway is essential for IL-1beta-induced ICAM-1 gene expression in A549 cells. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in the airway disease.     

ERK MAP kinase links cytokine signals to activation of latent HIV-1 infection by stimulating a cooperative interaction of AP-1 and NF-kappaB.

Human immunodeficiency virus type 1 (HIV-1) can establish latent infection following provirus integration into the host genome. NF-kappaB plays a critical role in activation of HIV-1 gene expression by cytokines and other stimuli, but the signal transduction pathways that regulate the switch from latent to productive infection have not been defined. Here, we show that ERK1/ERK2 mitogen-activated protein kinase (MAPK) plays a central role in linking signals at the cell surface to activation of HIV-1 gene expression in latently infected cells. MAPK was activated by cytokines and phorbol 12-myristate 13-acetate in latently infected U1 cells. The induction of HIV-1 expression by these stimuli was inhibited by PD98059 and U0126, which are specific inhibitors of MAPK activation. Studies using constitutively active MEK or Raf kinase mutants demonstrated that MAPK activates the HIV-1 long terminal repeat (LTR) through the NF-kappaB sites. Most HIV-1 inducers activated NF-kappaB via a MAPK-independent pathway, indicating that activation of NF-kappaB is not sufficient to explain the activation of HIV-1 gene expression by MAPK. In contrast, all of the stimuli activated AP-1 via a MAPK-dependent pathway. NF-kappaB and AP-1 components c-Fos and c-Jun were shown to physically associate by yeast two-hybrid assays and electrophoretic mobility shift assays. Coexpression of NF-kappaB and c-Fos or c-Jun synergistically transactivated the HIV-1 LTR through the NF-kappaB sites. These studies suggest that MAPK acts by stimulating AP-1 and a subsequent physical and functional interaction of AP-1 with NF-kappaB, resulting in a complex that synergistically transactivates the HIV-1 LTR. These results define a mechanism for signal-dependent activation of HIV-1 replication in latently infected cells and suggest potential therapeutic strategies for unmasking latent reservoirs of HIV-1.     

Activated human Langerhans cells express mRNA for IL-1 alpha and IL-1 beta and produce these cytokines but do not secrete them.

Human Langerhans cells (LC) were isolated from epidermal cell preparations by panning with mouse anti-CD1 monoclonal antibody. RNA was prepared and probed for the presence of mRNAs for various cytokines using radiolabeled cDNAs. After stimulation with phorbol myristate acetate LC express RNA for interleukin 1 alpha (IL-1 alpha) and interleukin 1 beta (IL-1 beta) and produce proteins but do not secrete them at detectable levels. LC-associated IL-1, particularly IL-1 alpha, may play a role in antigen presentation. PMA did not induce IL-6 expression in LC. The addition of lipopolysaccharide, a muramyl dipeptide analog, ionomycin, IL-1 alpha, tumor necrosis factor-alpha, insulin-like growth factor-1 or IL-6 did not induce IL-1 mRNA in LC. UVB augmented IL-1 beta mRNA expression. Glucocorticoids did not detectably affect IL-1 alpha or IL-1 beta mRNA levels following PMA induction, however, staurosporin inhibited IL-1 beta mRNA synthesis. Thus the inducers and regulators of IL-1 formation in human LC and monocytes are not identical.     

Cell-specific differences in activation of NF-kappa B regulatory elements of human immunodeficiency virus and beta interferon promoters by tumor necrosis factor.

Three aspects of the involvement of tumor necrosis factor in human immunodeficiency virus (HIV) pathogenesis were examined. Tumor necrosis factor alpha (TNF-alpha) mRNA production was analyzed by polymerase chain reaction amplification in monocytic U937 cells and in a chronically HIV infected U937 cell line (U9-IIIB). TNF-alpha RNA was undetectable in U937 cells, whereas a low constitutive level was detected in U9-IIIB cells. Paramyxovirus infection induced a 5- to 10-fold increase in the steady-state level of TNF-alpha RNA in U9-IIIB cells compared with U937 cells, suggesting that HIV-infected monocytic cells produced higher levels of TNF-alpha than did normal cells after a secondary virus infection. The effects of TNF-alpha on gene expression were examined by transient expression assays using reporter chloramphenicol acetyltransferase plasmids linked to regulatory elements from the HIV long terminal repeat (LTR) and the beta interferon promoter. In U937 and Jurkat T lymphoid cells, the inducibility of the different hybrid promoters by TNF-alpha or phorbol ester varied in a cell type- and promoter context-specific manner; the levels of gene activity of NF-kappa B-containing plasmids correlated directly with induction of NF-kappa B DNA-binding activity. Although the intact beta interferon promoter was only weakly stimulated by phorbol ester or TNF-alpha, multimers of the PRDII NF-kappa B-binding domain were inducible by both agents. TNF-alpha was able to increase expression of the HIV LTR in T cells, but in monocytic cells, TNF-alpha did not induce the HIV LTR above a constitutive level of activity. This level of NF-kappa B-independent activity appears to be sufficient for virus multiplication, since TNF-alpha treatment had no effect on the kinetics of de novo HIV type 1 (HIV-1) infection and viral RNA production in U937 cells. However, in Jurkat cells, TNF-alpha dramatically enhanced the spread of HIV-1 through the cell population and increased viral RNA synthesis, indicating that in T cells HIV-1 multiplication was stimulated by TNF-alpha treatment.     

TNF-α-mediated activation of HIV-1 LTR in monocytoid cells by mycobacteria

Mycobacterial infection occurs commonly in patients with acquired immune deficiency syndrome. Incubation of monocytoid cell line U937 cells, which was cotransfected HIV-1 long terminal repeat sequence (LTR) chloramphenicol acetyltransferase (CAT) plasmid and Tat expression plasmid, with Mycobacterium smegmatis, Mycobacterium avium, Mycobacterium bovis BCG and Mycobacterium tuberculosis resulted in enhancement of CAT production, indicating that these mycobacteria could activate LTR in this cell line. The amount of CAT in the cells coexisting with M. smegmatis was higher than that infected with other mycobacteria. The amounts of CAT production in the cells coculturing with M. avium and M. bovis BCG were intermediate. M. tuberculosis slightly stimulated CAT production. The amount of tumor necrosis factor (TNF)-alpha produced by transfected U937 cells was correlated with the amount of CAT production. The interleukin (IL)-1beta and IL-6 levels in the supernatant from coculturing with all species were similar. The antibody to TNF-alpha inhibited CAT production induced by mycobacterial infections. The anti-IL-1beta and anti-IL-6 antibodies, however, scarcely influenced stimulation of LTR by mycobacteria. In addition, U937 cells transfected with full length LTR CAT plasmid showed increased CAT production by activation with mycobacteria, but the cells transfected with mutant LTR CAT constructs from which the nuclear factor (NF)-kappaB binding site was deleted did not show activation. These findings indicated that activation of Mycobacterium-induced LTR CAT is NF-kappaB dependent. These findings suggested that activation of HIV-1 LTR by mycobacteria was mainly mediated by NF-kappaB-induced secondary release of cytokine TNF-alpha.     

Action of intracellular IL-1Ra (Type 1) is independent of the IL-1 intracellular signalling pathway

The balance between IL-1 and its naturally occurring inhibitor IL-1 receptor antagonist (IL-1ra) is critical in determining the inflammatory response. Four splice variants of the IL-1ra gene have been identified; one secreted (sIL-1ra) and three intracellular (icIL-1ra1-3). The biological roles of the intracellular isoforms remain largely unclear. We wished to determine whether icIL-1ra1 had intracellular functions regulating IL-1 signalling. Signalling was determined using an NF-kappaB reporter assay measuring induction of the IL-8 promoter in transfected cells. Over-expression of icIL-1ra1 in HeLa cells had no effect on IL-1 stimulated IL-8 activity. In contrast over-expression of sIL-ra significantly attenuated IL-1 activity. In addition, transfection of icIL-1ra1 in HeLa cells did not cause inhibition of IL-8 promoter activity following over-expression of the IL-1 signalling components MyD88, IRAK-1, TRAF-6, Ikappakappabeta or RelA. This implies that icIL-1ra1 does not act to alter IL-1 mediated intracellular signalling in this system. We investigated whether ATP and/or over-expression of the P2X7 receptor caused icIL-1ra1 inhibition of IL-1beta mediated IL-8 reporter activation, by permitting its release. In HeLa cells, no effect of icIL-1ra1 was observed in ATP stimulated and/or P2X7 transfected cells, compared to a significant inhibition in sIL-1ra transfected cells. However, in endothelial cells stimulated with ATP, the released fraction was effective in attenuating IL-1beta activation of the IL-8 reporter. These results suggest that icIL-1ra1 does not act at an intracellular level to alter IL-1 mediated signalling, and is effective in inhibiting IL-1 responses only when released in an ATP-dependent and cell type specific manner.     

Involvement of p42/p44 MAPK, p38 MAPK, JNK, and NF-kappaB in IL-1beta-induced VCAM-1 expression in human tracheal smooth muscle cells

Interleukin-1beta (IL-1beta) has been shown to induce the expression of adhesion molecules on airway epithelial and smooth cells and contributes to inflammatory responses. Here, the roles of mitogen-activated protein kinases (MAPKs) and nuclear factor-kappaB (NF-kappaB) pathways for IL-1beta-induced vascular cell adhesion molecule (VCAM)-1 expression were investigated in human tracheal smooth muscle cells (HTSMC). IL-1beta induced expression of VCAM-1 protein and mRNA in a time-dependent manner, which was significantly inhibited by inhibitors of MEK1/2 (U0126 and PD-98059), p38 (SB-202190), and c-Jun NH(2)-terminal kinase (JNK; SP-600125). Consistently, IL-1beta-stimulated phosphorylation of p42/p44 MAPK, p38, and JNK was attenuated by pretreatment with U0126, SB-202190, or SP-600125, respectively. IL-1beta-induced VCAM-1 expression was significantly blocked by the specific NF-kappaB inhibitors helenalin and pyrrolidine dithiocarbamate. As expected, IL-1beta-stimulated translocation of NF-kappaB into the nucleus and degradation of IkappaB-alpha were blocked by helenalin but not by U0126, SB-202190, or SP-600125. Moreover, the resultant enhancement of VCAM-1 expression increased the adhesion of polymorphonuclear cells to a monolayer of HTSMC, which was blocked by pretreatment with helenalin, U0126, SB-202190, or SP-600125 before IL-1beta exposure or by anti-VCAM-1 antibody. Together, these results suggest that in HTSMC, activation of p42/p44 MAPK, p38, JNK, and NF-kappaB pathways is essential for IL-1beta-induced VCAM-1 gene expression. These results provide new insight into the mechanisms of IL-1beta action that cytokines may promote inflammatory responses in airway disease.     

Mechanisms for macrophage-mediated HIV-1 induction

Viral latency is a long-term pathogenic condition in patients infected with HIV-1. Low but sustained virus replication in chronically infected cells can be activated by stimulation with proinflammatory cytokines such as TNF-alpha, IL-1 beta, or other host factors. However, the precise mechanism by which cellular activation induces latently infected cells to produce virions has remained unclear. In the present report, we present evidence that activation of HIV-1 replication in latently infected U1 or ACH2 cells by human macrophages is mediated by a rapid nuclear localization of NF-kappaB p50/p65 dimer with concomitant increased expression of proinflammatory cytokines. Multiplexed RT-PCR amplification of mRNA isolated from cocultures of macrophages and U1 and ACH2 cells showed significant induction of IL-1beta, IL-6, IL-8, TNF-alpha, and TGF-beta expression within 3 h of coincubation. Fixation of macrophages, U-1, or ACH2 cells with paraformaldehyde before coculture completely abrogated the induction of NF-kappaB subunits and HIV-1 replication, suggesting that cooperative interaction between the two cell types is an essential process for cellular activation. Pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with neutralizing anti-TNF-alpha Ab down-regulated the replication of HIV-1. In addition, pretreatment of macrophage-U1 or macrophage-ACH2 cocultures with the NF-kappaB inhibitor (E)3-[(4-methylphenyl)sulfonyl]-2-propenenitrile (BAY 11-7082) prevented the induction of cytokine expression, indicating a pivotal role of NF-kappaB-mediated signaling in the reactivation of HIV-1 in latently infected cells by macrophages. These results provide a mechanism by which macrophages induce HIV-1 replication in latently infected cells.     

Transactivation of human immunodeficiency virus type 1 long terminal repeats by cell surface tumor necrosis factor alpha.

Tumor necrosis factor alpha (TNF-alpha) is expressed in secreted and cell surface (csTNF-alpha) forms by activated monocytic and T cells. In this report, we demonstrate that csTNF-alpha may predominantly regulate the human immunodeficiency virus type 1 (HIV-1) long terminal repeat (LTR) activation in the promonocytic cell line U937 and in the Epstein-Barr virus-transformed B-cell line BH1. Anti-TNF-alpha antibody suppressed both the constitutive expression of the HIV-1 LTR in BH1 cells and the expression induced by phorbol 12-myristate 13-acetate in U937 cells. This suppression was found to be mediated via csTNF-alpha. No correlation between the HIV-1 LTR activation and the secretion of TNF-alpha was evident in these cell lines. Suppression of TNF-alpha secretion by cyclosporin A or by a serine protease inhibitor did not suppress the HIV-1 LTR activation. These observations suggest a novel biological role for csTNF-alpha in the immunopathogenesis of AIDS.     

Interleukin 1 induces HIV-1 expression in chronically infected U1 cells: blockade by interleukin 1 receptor antagonist and tumor necrosis factor binding protein type 1.

BACKGROUND: Cytokines and cytokine antagonists modulate human immunodeficiency virus (HIV) replication in vitro and may be involved in HIV disease pathogenesis. An understanding of these cytokine networks may suggest novel treatment strategies for HIV-seropositive persons. MATERIALS AND METHODS: U1 cells, a chronically infected promonocytic cell line, were stimulated with interleukin 1 alpha (IL-1 alpha), IL-1 beta or tumor necrosis factor (TNF) for 24 hr. The effects of these cytokines, and of anti-IL-1 receptor type 1 and type 2 (IL-1RI and II) antibody, IL-1 receptor antagonist (IL-1Ra), and recombinant human TNF binding protein type 1 (rhTBP-1, a form of TNF receptor p55), on HIV-1 replication, as measured by ELISA for HIV-1 p24 antigen, were determined. The effects of IL-1 and IL-1Ra on nuclear factor-kappa B (NF-kappa B) DNA binding activity, as measured by electrophoretic mobility shift assays, were also determined. RESULTS: IL-1 alpha and IL-1 beta increased p24 antigen production in a concentration-dependent manner. IL-1Ra completely, and rhTBP-1 partially, suppressed IL-1-induced p24 antigen production. IL-1 increased NF-kappa B DNA binding activity and IL-1Ra blocked this effect. Since IL-1Ra blocks IL-1 from binding to both the IL-1RI and Il-1RII, monoclonal antibodies directed against each receptor were used to ascertain which IL-1R mediates IL-1-induced HIV-1 expression. Antibody to the IL-1RI reduced IL-1-induced p24 antigen production. Although anti-IL-1RII antibody blocked the binding of 125IL-1-1 alpha to U1 cells by 99%, this antibody did not affect IL-1-induced p24 antigen production. IL-1 beta enhanced TNF alpha-induced HIV expression when added before or simultaneously with TNF alpha. CONCLUSIONS: IL-1 induces HIV-1 expression (via the IL-1RI) and NF-kappa B activity in U1 cells. These effects are blocked by IL-1Ra and partially mediated by TNF. IL-1 enhances TNF alpha-induced HIV replication in U1 cells.     

Regulation of granulocyte-macrophage colony-stimulating factor in human retinal pigment epithelial cells by IL-1beta and IFN-gamma.

GM-CSF production by RPE cells, which form part of the blood-retina barrier, is upregulated by IL-1beta and this increase can be reversed by IFN-gamma. IL-1beta up-regulation is not dependent on PKC but the PKC activator PMA induces low levels of GM-CSF production and acts synergistically with IL-1beta to further increase GM-CSF. Although A23187 and ionomycin stimulated low levels of GM-CSF production, the IL-1beta pathway was cyclosporin A insensitive and did not interact with the calcium pathway. IL-1beta-stimulated GM-CSF mRNA expression and production was strongly dependent on NF-kappaB. IFN-gamma inhibition of the GM-CSF response to IL-1beta acted via NF-kappaB, reducing the translocation of NF-kappaB to the nuclei of RPE cells treated with IL-1beta and IFN-gamma. The results show that IFN-gamma down-regulation acts either directly on NF-kappaB or its activation or by blockade of a pathway upstream of NF-kappaB. However, any such blockade does not involve PKC or intracellular calcium.     

Increased human immunodeficiency virus (HIV) expression in chronically infected U937 cells upon in vitro differentiation by hydroxyvitamin D3: roles of interferon and tumor necrosis factor in regulation of HIV production.

We have investigated the roles of cytokines in the modulation of human immunodeficiency virus (HIV) production in chronically infected U937 cells upon in vitro differentiation by hydroxyvitamin D3. HIV-infected U937 cells exhibited markedly lower levels of CD4 and HLA-DR antigens than uninfected cells did. Vitamin D3 induced a time-dependent macrophagelike differentiation, as determined by monitoring the expression of some surface antigens by means of the monoclonal antibodies OKM1, OKM5, OKM13, OKM14, OKT4, anti-HLA-DR, TecMG2, TecMG3, LeuM3, LeuM1, anti-HLA-DP, and anti-HLA-DQ. Treatment with hydroxyvitamin D3 resulted in a marked increase in HIV production compared with control cultures. Interleukin 1 beta (IL-1 beta) and tumor necrosis factor alpha (TNF-alpha) were detected in the culture media, whereas interferon (IFN) was not generally found. Using the polymerase chain reaction technique, we found HIV-infected U937 cells to express detectable levels of mRNAs for alpha interferon (IFN-alpha), IFN-beta, TNF-alpha, and IL-1 beta. The addition of TNF resulted in a marked increase of HIV production, whereas IL-1 beta was ineffective. In contrast, both IFN-alpha and IFN-beta exerted some inhibitory effect on HIV production, which was more marked in vitamin D3-treated cultures than in untreated cultures. HIV production was significantly increased by antibodies to IFN-alpha in both untreated and vitamin D3-treated cultures. Anti-IFN-beta antibody increased HIV production only in vitamin D3-treated cells. In contrast, anti-TNF-alpha antibodies markedly decreased HIV production in both control and differentiating U937 cells. Vitamin D3 treatment resulted in a higher expression of TNF receptors in differentiating cells than in control HIV-infected cells. These data demonstrate a strong correlation between HIV production and macrophagelike differentiation in chronically infected U937 cells and suggest that endogenous IFN and TNF exert opposite effects in the regulation of virus production in both undifferentiated and vitamin D3-treated cell cultures.