Trypanocidal drug benznidazole impairs lipopolysaccharide induction of macrophage nitric oxide synthase gene transcription through inhibition of NF-kappaB activation.

In murine macrophages, inducible NO synthase II (NOSII) gene expression is promoted at a transcriptional level by LPS and/or IFN-gamma with benznidazole (BZL), a trypanocidal drug, acting to down-regulate NOSII gene induction and hence inhibiting NO production. By performing transient transfection experiments, we now report that BZL also inhibited the expression of NOSII gene promoter or multimerized NF-kappaB binding site controlled reporter genes. By contrast, no effect was observed on the expression of a reporter gene under the control of the NOSII promoter-derived IFN regulatory factor element. EMSAs demonstrated that BZL inhibited the nuclear availability of NF-kappaB in stimulated macrophages. NF-kappaB is activated in macrophages by phosphorylation, ubiquitination, and subsequent proteolysis of IkappaB. Within this setting, Western blot was also performed to show that BZL blocked IkappaBalpha degradation. Collectively, these results demonstrate that BZL is able to specifically inhibit macrophage NF-kappaB activation after LPS plus IFN-gamma stimulation.

IFN-beta induces serine phosphorylation of Stat-1 in Ewing's sarcoma cells and mediates apoptosis via induction of IRF-1 and activation of caspase-7.

Four human cell lines derived from Ewing's sarcoma, EW-7, EW-1, COH and ORS, were investigated to establish the effects of human recombinant interferon-alpha2a and human recombinant interferon-beta on cell proliferation and apoptosis. All four cell lines were much more sensitive to the antiproliferative effects of IFN-beta than of IFN-alpha. Analysis of the early signals triggered by IFN-alpha and IFN-beta demonstrated that the two IFNs were similarly effective in inducing tyrosine phosphorylation of the Jak-1 and Tyk-2 kinases and the transcription factors Stat-1 and Stat-2. Interestingly, an additional rapid phosphorylation of Stat-1 on serine was observed after IFN-beta treatment, with concomitant activation of p38 mitogen-activated protein kinase. In these cells, Stat-1 Ser727 phosphorylation in response to IFN-beta was found to be impaired by p38 MAPkinase inhibitor (SB203580). IFN-beta induced the formation of the Interferon Stimulated Gene Factor 3 complex more efficiently than IFN-alpha, as well as sustained induction of IRF-1, which may account for its greater induction of 2'5'oligo(A)synthetase and greater inhibition of cell proliferation. IFN-beta, but not IFN-alpha, induced apoptosis in wild-type p53 EW-7 and COH cell lines, but not in the mutated p53 EW-1 or ORS cell lines. The apoptosis induced by IFN-beta in EW-7 and COH cell lines appeared to be mediated by IRF-1 and involved the activation of caspase-7. Ectopic expression of IRF-1 induced apoptosis in all four cell lines which correlated with the activation of caspase-7 and with the downregulation of the Bcl-2 oncoprotein, as observed for IFN-beta-induced apoptosis in parental EW-7 and COH cell lines.

Production of nitric oxide (NO) in human hydatidosis: relationship between nitrite production and interferon-gamma levels.

Human hydatidosis is characterized by a prolonged coexistence of parasite (Echinococcus granulosus) and host without effective rejection. The basis of the immune response of the patient is poorly understood. Previously, we reported the presence of IFN, TNF-alpha and IL-6 activities in the serum of patients with liver and lung hydatidosis. In the present work, we have investigated the production of nitrite (NO2-) in the serum of hydatidic patients carrying hepatic and pulmonary cysts (range 36-300 microM). Our present data show a correlation between the production of nitrite + nitrate (NO2- + NO3-) and that of circulating cytokines IFN and IL-6. In relapsing patients who did not produce IFN and IL-6, the observed serum NO2- concentrations were low (range 10-37.2 microM), as compared to those detected in patients before surgery. Induction of NO synthase in leukocytes from hydatidic patients was induced by stimulating these cells with a specific parasitic antigen, Antigen-5, as assessed by the increased levels of NO3- + NO2- in the range of 60-85 microM for patients with liver hydatidosis, as compared to the 20-25 microM detected in healthy controls. Collectively, our data indicate that NO2- + NO3- levels correlate with IFN levels and immunoreactivity, and overall suggest that IFN-gamma and nitric oxide production together play a role in the host defense mechanisms in human hydatidosis.

Inhibitors of ADP-ribosylation impair inducible nitric oxide synthase gene transcription through inhibition of NF kappa B activation.

In murine macrophages, inducible NO synthase II (iNOS or NOS-II) is induced at the transcriptional level by IFN-gamma, alone or synergistically with LPS. We investigated the possible role of reactions of ADP-ribosylation in triggering the signaling pathways involved in NOS-II gene expression. Stimulation with IFN-gamma and/or LPS of RAW 264.7 macrophages, transiently transfected with the NOS-II promoter, was inhibited by ADP-ribosylation inhibitors, indicating that they interfered with the signal(s) responsible for NOS-II gene transcription. We therefore explored the effect of these inhibitors on the activity of IRF-1 and NF kappa B transcription factors known to be involved in NOS-II induction by IFN-gamma and LPS. No effect was observed on IRF-1 activation. However, NF kappa B binding to its target sequence diminished and transfection experiments with an NF kappa B-driven reporter plasmid demonstrating that ADP-ribosylation inhibitors suppressed NF kappa B-dependent promoter activity. These results provide evidence that a step involving ADP-ribosylation is required to activate NF kappa B-mediated gene transcription.

Relationship among circulating interferon, tumor necrosis factor-alpha, and interleukin-6 and serologic reaction against parasitic antigen in human hydatidosis.

Human hydatidosis is a parasitic disease vectored by the larval stage cestode Echinoccocus granulosus. It constitutes a major health problem in North Africa. We investigated the production of circulating interferon (IFN), tumor necrosis factor-alpha (TNF-alpha), and interleukin-6 (IL-6) in Algerian patients with liver, lung, or ocular hydatidosis. In all, 101 serum samples from these patients with analyzed. Immunoreactivity and cytokine activities were undetectable in sera from ocular hydatidosis patients. However, we observed the presence of IFN (a mixture of IFN-alpha, IFN-beta, and IFN-gamma, range 32-500 U/ml), TNF-alpha (range 32-100 U/ml), and IL-6 (range 32-500 U/ml) in all patients who had liver or lung cysts or both and displayed immunoreactivity against parasitic antigen (antigen 5). After surgical removal of the cysts, serum cytokine levels declined rapidly and were undetectable at 30 days. IFN and IL-6 activity was undetectable in sera from two liver hydatidosis patients who relapsed and did not display any immune response against parasitic antigen. These results suggest that in liver and lung hydatidosis, cytokine production contributes to the host defense mechanism against the extracellular parasite.

Differential expression of inducible NO synthase in two murine macrophage cell lines.

Although primary macrophages and most murine macrophage cell lines such as RAW 264.7 cells respond to interferon-gamma (IFN-gamma) and/or lipopolysaccharide (LPS) by producing large amounts of nitrite, i.e. the oxidation product of nitric oxide (NO) produced by inducible NO synthase (iNOS), other cell lines like P388.D1 cells do not produce significant amounts. To gain insight into the signalling pathway that leads to the induction of iNOS activity, we compared iNOS expression in RAW 264.7 and P388.D1 cells. We showed that IFN-gamma binds to each cell line with a similar affinity. Furthermore, no differences in iNOS gene structure were detectable by Southern blot analysis. Even though no significant nitrite secretion was found in the supernatant of P388:D1 cells stimulated with IFN-gamma and/or LPS, iNOS mRNA expression was induced. In addition, IFN-gamma induced the interferon regulatory factor-1 (IRF-1) gene and activated the binding of this factor to its target sequence in the iNOS gene. This binding was recently shown to be necessary for iNOS expression. However, in P388.D1 cells, we were unable to detect the corresponding iNOS protein. These results indicate a deficiency in P388.D1 cells which appears to be restricted to the signalling pathway controlling iNOS protein synthesis. This deficiency does not affect the overall IFN-gamma biological response, but rather a convergent post-transcriptional step common to IFN-gamma and LPS.

Triggering of the human interleukin-6 gene by interferon-gamma and tumor necrosis factor-alpha in monocytic cells involves cooperation between interferon regulatory factor-1, NF kappa B, and Sp1 transcription factors.

We investigated the molecular basis of the synergistic induction by interferon-gamma (IFN-gamma)/tumor necrosis factor-alpha (TNF-alpha) of human interleukin-6 (IL-6) gene in THP-1 monocytic cells, and compared it with the basis of this induction by lipopolysaccharide (LPS). Functional studies with IL-6 promoter demonstrated that three regions are the targets of the IFN-gamma and/or TNF-alpha action, whereas only one of these regions seemed to be implicated in LPS activation. The three regions concerned are: 1) a region between -73 and -36, which is the minimal element inducible by LPS or TNF-alpha; 2) an element located between -181 and -73, which appeared to regulate the response to IFN-gamma and TNF-alpha negatively; and 3) a distal element upstream of -224, which was inducible by IFN-gamma alone. LPS signaling was found to involve NF kappa B activation by the p50/p65 heterodimers. Synergistic induction of the IL-6 gene by IFN-gamma and TNF-alpha, in monocytic cells, involved cooperation between the IRF-1 and NF kappa B p65 homodimers with concomitant removal of the negative effect of the retinoblastoma control element present in the IL-6 promoter. This removal occurred by activation of the constitutive Sp1 factor, whose increased binding activity and phosphorylation were mediated by IFN-gamma.

Abnormal lymphokine production: a novel feature of the genetic disease Fanconi anemia. II. In vitro and in vivo spontaneous overproduction of tumor necrosis factor alpha.

We have previously shown an unbalanced cytokine production in Fanconi anemia (FA) cells, ie, an underproduction of interleukin 6 (IL-6) during growth. Among a number of cytokines analyzed, the only other anomalies detected concern tumor necrosis factor alpha (TNF alpha). In comparison to normal cells, this cytokine is overproduced by FA lymphoblasts from the four genetic complementation groups. Indeed, up to an eight-fold increase in TNF alpha is observed in the growth medium of FA cells. Moreover, addition of anti-TNF alpha antibodies partially corrects the FA hypersensitivity to treatment by mitomycin C (MMC). Treatment of FA cells with IL-6, which partially restored an almost normal sensitivity to MMC of FA cells also reduces the TNF alpha overproduction in FA lymphoblasts. No anomalies at the molecular level (Southern and Northern blot analyses) are detected for the TNF alpha gene and its mRNA. We have investigated the in vivo situation by assaying TNF alpha levels in the serum from FA homozygotes and obligate heterozygotes. In contrast to normal healthy donors or to aplastic anemia patients in whom serum TNF alpha is present only in trace amounts, all 36 FA patients and 21 FA parents monitored show a significantly (P < .001) higher level of serum TNF alpha activity. Consequently, abnormal TNF alpha production seems to be associated with the FA genetic background.

Inactivation of interleukin-6 in vitro by monoblastic U937 cell plasma membranes involves both protease and peptidyl-transferase activities.

Human promonocytic U937 cells have previously been shown to possess at their cell surface specific transmembrane serine proteases and N-terminal amino acid proteases as well as associated enzymes including elastase and cathepsin G. In this study, purified plasma membranes from U937 cells are reported to degrade the recombinant 21-kDa 125I-interleukin-6 (125I-IL-6) into 8-kDa products with loss of biological activity, as monitored by polyacrylamide gel electrophoresis and a cell-proliferation bioassay. Degradation of 125I-IL-6 by plasma membranes was completely prevented by the serine-protease inhibitor diisopropyl fluorophosphate, but was only partially impaired by alpha 1-protease inhibitor and antibody against cathepsin G. A similar incubation of 125I-IL-6 with cathepsin G purified from U937 cells caused hydrolysis of the cytokine into similar inactive 8-kDa fragments, whereas incubation with purified U937 cell elastase failed to degrade the peptide. These findings indicate that U937 cells hydrolyze IL-6 using cell-associated serine-protease activity and that cathepsin G partially participates in this degradation. Prolonged incubation of 8-kDa 125I-IL-6 fragments with purified U937 plasma membranes, led to a complete loss of IL-6 activity related to the transformation of the 8-kDa forms into a higher-molecular-mass complex (16 kDa). This complex was stable in SDS and 2-mercaptoethanol at 100 degrees C and was not dissociated by hydroxylamine treatment, indicating the formation of a covalent non-ester bond between the 8-kDa 125I-IL-6-derived peptide and an undetermined acceptor. An initial oxidative treatment of 125I-IL-6 partially prevented complex formation, suggesting the presence of one or more oxidizable methionine residues at the binding site of 8-kDa 125I-IL-6 peptide. The kinetics of complex formation (time dependence and plasma-membrane-concentration dependence), as well as its inhibition by a specific inhibitor of N-amino-peptidase activity, bestatin, suggest the participation of peptidyl-transferase activity in complex formation. Finally, a plasma-membrane fraction, corresponding to a molecular mass > or = 30 kDa, was able to convert the 8-kDa 125I-IL-6 forms into the 125I-labeled 16-kDa complex, suggesting that a > or = 30-kDa peptidyl-transferase enzyme catalyzes the reaction and provides the 125I-labeled 16-kDa peptide by dimerization of 8-kDa 125I-IL-6-derived intermediates. Further identification of the plasma-membrane-associated peptidyl transferase as a regulator of IL-6 proteolysis may be of physiological relevance for the control of IL-6 biological activity.

Interferon-gamma modulates retinoblastoma gene mRNA in monocytoid cells.

To study the effect of interferon gamma (IFN-gamma) on the expression of the retinoblastoma (RB) susceptibility gene, we performed Northern-blot analysis on RNA extracted from Wish, HEL and monocytoid cell lines U-937 and THP-1 treated with 1,000 IU/ml of recombinant IFN-gamma. In U-937 and THP-1 cells, IFN-gamma increased the abundance of RB mRNA. In Wish and HEL cells, co-treatment with cycloheximide was required for IFN-gamma to increase the level of RB mRNA. Pre-treatment of THP-1 cells with cycloheximide prior to IFN-gamma treatment augmented the effects of IFN-gamma on RB gene expression. The effect of IFN-gamma in THP-1 cells was observed after 3 hr of treatment, being more pronounced after 6 hr and persisting until at least 18 hr, although at a lower level. These results suggest that IFN-gamma regulates the level of RB mRNA by different mechanisms in the different cell types. This cytokine increases the abundance of RB mRNA in monocytoid cell lines, reinforced by prior treatment with cycloheximide. Inhibition of protein synthesis is required in Wish and HEL cell lines before IFN-gamma has an effect on RB gene expression.

Tumor necrosis factor-alpha and IL-6 up-regulate IFN-gamma receptor gene expression in human monocytic THP-1 cells by transcriptional and post-transcriptional mechanisms.

IFN-gamma is a potent activator of monocytic cell functions, including the stimulation of TNF-alpha and the control of IL-6 synthesis. These cytokines might act as autocrine or paracrine factors together with IFN-gamma in inducing biologic responses. In this study, we addressed the question of whether or not TNF-alpha and IL-6 play a role in modulating of IFN-gamma binding. Expression of IFN-gamma surface receptor was measured in the human monocytic THP-1 cells. The capacity of these cells to bind IFN-gamma increased with time after treatment with each cytokine. The number of IFN-gamma R per cell rose by three- and fourfold after 16 h of treatment with TNF-alpha and IL-6, respectively. Scatchard analysis of binding data showed that neither TNF-alpha nor IL-6 affected the affinity of IFN-gamma for its receptor. The increased surface expression of IFN-gamma R induced by TNF-alpha and IL-6 correlated with the rise in IFN-gamma mRNA receptor levels. TNF-alpha-mediated up-regulation of IFN-gamma R was due to an increase in transcriptional activity of the IFN-gamma R gene, as shown by run-on experiments. No significant modulation of IFN-gamma R gene transcription was observed in nuclei from cells treated with IL-6, whose effect appeared to be related to IFN-gamma mRNA receptor stabilization. Taken together, the present results provide, to our knowledge, the first evidence for cytokine-mediated up-regulation of IFN-gamma R in human monocytic cells. This up-regulation by TNF-alpha and IL-6, which are both produced by monocytes, occurs through different mechanisms, and may be of physiologic relevance in host defense.

IFN-gamma and transforming growth factor-beta 1 differently regulate fibronectin and laminin receptors of human differentiating monocytic cells.

The regulation of extracellular matrix (ECM) protein receptor expression was followed in the human promonocytic cell line U937 before and after stimulation either with PMA or various cytokines implicated in monocytopoiesis. On undifferentiated U937 cells, alpha-chains of very late Ag (VLA)-4, VLA-5, and VLA-6 were constitutively expressed whereas alpha-chains of VLA-2 (alpha 2) and vitronectin receptor (alpha V) were not. Maturation of U937 cells with PMA resulted in a marked decrease in alpha 4 expression (25% of control by day 5), and a small but significant increase in the expression of alpha 2 and alpha v over 4 days of stimulation. Unstimulated U937 cells attached to fibronectin (FN) but not to laminin (LM), collagens I/IV-coated surfaces. After PMA stimulation, U937 cells exhibited enhanced adherence on FN and expressed the ability to adhere to LM. PMA stimulation also promoted U937 spreading both on FN and LM. Adhesion on FN all along the maturation pathway was specifically and totally inhibited by anti-alpha 5 mAb but not by anti-alpha 4 mAb. Anti-beta 1, anti-alpha 6, anti-alpha 2, and anti-alpha v mAb, as well as Tyr-Ile-Gly-Ser-Arg and Arg-Gly-Asp synthetic peptides from LM, had no effect on adhesion of PMA-stimulated cells on LM, implying that U937 cell adherence to LM is mediated through hitherto distinct receptors. In the presence of rIFN-gamma, differentiating U937 cells did not adhere to LM and lost the capacity to bind to FN. Loss of adhesion to FN was correlated with the concomitant decrease in the expression of alpha 4 and alpha 5 integrin subunits. In contrast, TGF-beta 1 mimicked most of the effects of PMA by enhancing the attachment of maturating U937 cells on FN through alpha 5 receptors and by promoting adherence to LM. TGF-beta 1 stimulation also promoted U937 cell spreading on both FN- and LM-coated surfaces. The data suggest that inflammatory cytokines such as IFN-gamma and TGF-beta 1 may be critically important in the homing of monocytic cells at sites of inflammation by modulating cell-surface expression of ECM receptors.

Pentoxifylline inhibits certain constitutive and tumor necrosis factor-alpha-induced activities of human normal dermal fibroblasts.

Pentoxifylline (PFN), analog of theobromine, which phenotypically and functionally alters various cell types including dermal fibroblasts, has been reported to inhibit tumor necrosis factor-alpha (TNF alpha) activation of neutrophils. We investigated the ability of PFN to alter constitutive and TNF alpha-induced biosynthetic activities of human normal dermal fibroblasts. The sixteenfold increase over constitutive intracellular 2'-5' oligo-adenylate synthetase (2'-5' A synthetase) activity induced by TNF alpha (400 U/ml) failed to occur when PFN (1 mg/ml) was added prior to cytokine treatment. This loss of biologic activity paralleled a reduction in 2'-5' A synthetase proteins and 2'-5' A synthetase-specific m-RNA. PFN failed to inhibit constitutive or TNF alpha-induced IL-6 hybridoma proliferative activity, IL-6 protein, or IL-6-specific m-RNA levels. The presence of PFN (1 mg/ml) in fibroblast cultures reduced constitutive synthesis of collagen and glycosaminoglycan (GAG) by 87% and 45%, respectively, and blocked induction of their synthesis by TNF alpha (10(4) U/ml). Total non-collagenous protein synthesis was not inhibited following PFN treatment (1 mg/ml). PFN did not inhibit TNF alpha induction of only those biosynthetic activities also susceptible to PFN in the constitutive state, with PFN failing to reduce constitutive collagenolytic activity but reducing TNF alpha-induced enhanced collagenolytic activity by 26% and collagenase m-RNA by 51%. Furthermore, PFN did inhibit, by 98%, TNF alpha-dependent murine and human fibroblast cytotoxicity. The selective nature of PFN inhibition of certain TNF alpha activities, the failure of PFN (1 mg/ml) to alter constitutive and TNF alpha-induced levels of type 1 and 2 TNF alpha receptor m-RNA, and the finding that PFN-treated fibroblasts express a similar number of receptors, of similar molecular weight and high affinity for TNF alpha as control, untreated cells, suggest that inhibitory activities of PFN are mediated at a locus other than receptors for TNF alpha.

Abnormal lymphokine production: a novel feature of the genetic disease Fanconi anemia. I. Involvement of interleukin-6.

The correction of chromosomal hypersensitivity to mitomycin C (MMC) in Fanconi anemia (FA) human lymphoblasts is observed by growth in a medium conditioned by normal human cells. Under the same conditions, the cytotoxic effect of MMC on FA cells is restored to an almost normal level. The addition of interleukin-6 (IL-6) to an unconditioned culture medium increased the resistance of FA cells to MMC cytotoxicity. This correcting effect is partially abolished by addition of an anti-IL-6 antibody to the conditioned medium. Both lymphoblasts and fibroblasts derived from FA patients demonstrate a reduction in IL-6 production. Moreover, this lymphokine is not induced by tumor necrosis factors alpha and beta (TNF alpha and TNF beta) in FA cells, as is the case in normal cells. It is suggested that the observed deficiency in IL-6 production may account for one of the major characteristics of FA disease, i.e., the defect in differentiation of the hematopoietic system.

Human U937 cell surface peptidase activities: characterization and degradative effect on tumor necrosis factor-alpha.

Surface peptidase activities on the human monocytic lineage cell line U937 were characterized. Two diisopropyl phosphofluoridate (DFP)-inhibitable serine peptidases were identified by differences in their hydrolytic activities on chromogenic peptides: one removed tripeptides from the free NH2-terminal end of the synthetic peptide Ala-Ala-Phe-p-nitroanilide (pNA) and was not inhibited by inhibitors of metallo-, cysteic-, and aspartic-proteinases, or by those of elastase-, trypsin- and chymotrypsin-like enzymes, suggesting the presence of a hitherto unidentified serine tripeptidyl endopeptidase; the other peptidase catalyzed the release of Gly-Pro from Gly-Pro-pNA and was inhibited by DFP, phenylmethyl sulfonyl fluoride and diprotin A, thus resembling dipeptidyl peptidase IV (DPP IV) with respect to its substrate specificity and inhibitor profile. A group of N-exo-aminopeptidase activities specifically inhibited by bestatin, was also detected when Ala-, Leu-, Arg- and Lys-pNA were used a substrates. The activities were surface associated and not secreted as determined by extracellular location of product and enzymatic recovery in highly purified U937 cell membranes. Peripheral monocytes and macrophages were found to virtually exhibit identical levels of these two classes of peptidase activities when compared to those detected on U937 cells. The relative contributions of these hydrolytic enzymes to the cleavage of bioactive and radioiodinated cytokines including tumor necrosis factor-alpha (TNF-alpha), interleukin-1 alpha and interferon-gamma was next examined. The results indicated that N-aminopeptidases do not appear to participate in the catabolism of any tested cytokine. In contrast, the most interesting finding was that both serine peptidases participate in TNF-alpha degradation. Analysis of the final proteolytic digestion products demonstrated the disappearance of the native 17-kDa molecule TNF-alpha, and the concomitant release of biologically inactive fragments of less than or equal to 2 kDa. Together, these observations indicate new roles for both the DPP IV-like enzyme and the tripeptidyl endopeptidase located at the surface of human monocytic cells, including the regulation of the extracellular TNF-alpha concentration. Thus, the identification of functional ectopeptidases provides insight into their potential role in both normal and malignant monocytic function.

Human endothelial cells transfected by SV40 T antigens: characterization and potential use as a source of normal endothelial factors.

A putative role for the vascular endothelium as target for autoantibodies has been suggested in several autoimmune disorders and connective-tissue diseases. However, there are some difficulties linked to the use of cultured endothelial cells (EC) that limit considerably the extensive studies on the nature of endothelial target antigens involved. To overcome this problem, human EC, derived from umbilical veins, were transfected with recombinant plasmid pSV1 which contained the early genes of simian virus SV40. These transfected cells, called EC-pSV1, are able to grow without EC growth supplement and demonstrate a population doubling time of about 50 h. Among the EC properties, EC-pSV1 retain intracellular content of angiotensin-converting enzyme activity, exhibit constitutive production of interleukin 6 and of a growth-promoting activity on early passage EV, express intercellular adhesion molecule 1 (ICAM-1) and its up-regulation by tumor necrosis factor alpha, but have lost the expression of factor VIII-related antigen. Moreover, EC-pSV1 express a 55-kDa antigen found on EC and human platelets, and presumably acting as an antibody target in some cases of non-allergic asthma. However, at the 50-55th generation, morphological changes and altered growth behavior were visible. This work demonstrates that transfection of EC with SV40 T antigens may be of interest, particularly in areas of research including the study of EC targets involved in different human diseases.

IL-6 and IL-6 receptor modulation by IFN-gamma and tumor necrosis factor-alpha in human monocytic cell line (THP-1). Priming effect of IFN-gamma.

The present work is a detailed study of the mechanism of IFN-gamma- and TNF-alpha-triggered IL-6 secretion and IL-6 gene expression in human monocytic THP-1 cells and of the effect of these cytokines on the expression of IL-6 surface receptor and IL-6R gene. Although TNF-alpha was shown to stimulate IL-6 expression in fibroblasts in monocytic THP-1 cells, IFN-gamma is required for TNF to induce IL-6 expression. The results reported here demonstrate that combined treatment of THP-1 cells with IFN-gamma + TNF-alpha induced IL-6 mRNA expression, whereas no significant induction was obtained by either cytokine alone. Nuclear run-on transcription assay showed that the increased level of IL-6 mRNA induced by IFN-gamma + TNF-alpha was associated with induction of gene transcription. Sequential stimulation of THP-1 cells by IFN-gamma and subsequently by TNF-alpha did not allow IL-6 gene induction, suggesting that IFN-gamma and TNF-alpha induced or activated different signaling factors which should act together to trigger IL-6 gene transcription. IFN-gamma pretreatment followed by IFN-gamma + TNF-alpha restimulation led to superinduction of the IL-6 gene expression with a concomitant increase in IL-6-secreted activity. This priming effect of IFN-gamma is dependent on active protein synthesis. Biochemical characterization of IL-6 proteins secreted by THP-1 cells by Western blotting and affinity chromatography allowed identification of a major IL-6 molecular species of 42 kDa and a minor one of 23 kDa. Furthermore, we showed here that IFN-gamma increased the IL-6R mRNA level with a concomitant increase in IL-6-specific binding to surface receptors. On the contrary, treatment with IFN-gamma + TNF-alpha reduced the level of IL-6R mRNA and IL-6 binding to THP-1 cells probably due to a ligand-mediated effect. Taken together, results reported here provide evidence that functional interaction between IFN-gamma and TNF-alpha is involved in the regulation of IL-6 and IL-6R expression in monocytic cells. Control of IL-6 production and IL-6R expression may be one of the important homeostatic properties of IFN-gamma.

Secretion of interleukin-6 (IL-6) by human monocytes stimulated by muramyl dipeptide and tumour necrosis factor alpha.

We studied IL-6 gene expression in human monocytes stimulated by muramyl dipeptide (MDP), a synthetic immunomodulator derived from mycobacterial cell walls. In control monocytes, two IL-6 transcripts of 3.4 kb and 1.6 kb were easily detected at 2.5 hr of culture and remained stable until 18 hr. In MDP-treated monocytes, three IL-6 RNA species displayed different kinetics of accumulation: a 3.4 kb RNA whose expression already reached its maximum after 2.5 hr exposure to MDP; a 1.6 kb RNA whose expression peaked at 5 hr; and a new RNA species of 1.4 kb which was transiently induced in early time of cell stimulation. TNF-alpha co-operated with MDP to increase IL-6 gene expression and secretion of biological active protein (measured by the hybridoma plasmacytoma growth factor assay). MDP exhibits a broad spectrum of immunomodulation properties such as adjuvant activity, enhancement of macrophage cytotoxicity against tumour and induction of non-specific resistance to intracellular agents. The results reported here suggest that these properties might be linked to the stimulation by MDP of genes coding for key cytokines such as IL-6, TNF and IL-1.