The Eft of Dual Inhibitor, Sk&F 86002, On Helper T Cell Functions

SK&F 86002, a mixed cyclooxygenase-lipoxygenase inhibitor, was examined for its effects on helper T cell functions. The drug was found to inhibit concanavalin A-induced mitogenesis of splenic T cells (IC₅₀ = 13 mμm), the mixed lymphocyte response (IC₅₀ = 16 mμM), and proliferation of antigen specific T cells (cloned line, IC₅₀ = 11 mμM; uncloned line, IC₅₀ = 13 mμM). In contrast, another mixed cycloxygenase-lipoxygenase inhibitor, BW775c, did not have such effects at non-cytotoxic levels. These T cell functions are believed to be dependent on the effects of elaborated IL-1. SK&F 86002 has been shown to inhibit the production of mature IL-1 (IC₅₀ = 1 mμM), possibly accounting for the anti-inflammatory effects of the drug in rheumatoid arthritis models. In an in vivo model of contact sensitivity, SK&F 86002 was able to inhibir mouse footpad swelling, demonstrating additional anti-inflammatory activity. As an inhibitor of IL-1 synthesis or release, SK&F 86002 may be useful for the treatment of T cell-dependent inflammation.     

Interleukin-10 regulates TNF-alpha-converting enzyme (TACE/ADAM-17) involving a TIMP-3 dependent and independent mechanism

IL-10 is a potent anti-inflammatory molecule, which regulates TNF-alpha at multiple levels. We investigated whether IL-10 also modulated the activity of the TNF-alpha-converting enzyme (TACE). Using an ex vivo fluorogenic assay we observed that LPS rapidly induced TACE activity in monocytes coinciding with release of soluble TNF-alpha. In the presence of IL-10, TNF-alpha production and activation of surface TACE was significantly inhibited. Paradoxically, both LPS with or without IL-10 led to accumulation of surface TACE (albeit catalytically inactive) over a 24 h period. We investigated whether this was mediated through induction of endogenous tissue inhibitor metalloproteinase-3 (TIMP-3). We found that the inhibition of TACE activity at 2 h by IL-10 was not TIMP-3 dependent but that the late accumulation of surface TACE was prevented with TIMP-3 antibodies. Furthermore, induction of endogenous TIMP-3 was observed by western blotting in both LPS- and in LPS with IL-10-treated monocytes from 6 to 8 h of culture. These results indicate that IL-10 further regulates TNF-alpha by modulating TACE activation at early time points and by contributing to the induction of TIMP-3, the natural inhibitor of active TACE, at later time points. These observations add to our understanding of inflammation and the importance of homeostatic regulators of these events.     

Superoxide Dismutase Specifically Inhibits Erythroid Cell DNA Synthesis and Proliferation

Abstract

The antioxidant enzyme superoxide dismutase (SOD) was previously shown to inhibit both the proliferation of murine erythroid DA-1 cells growing in the presence of Interleukin-3 (IL-3) and the DNA synthesis of marrow erythroid progenitor cells (BFU-E) in vitro. We show here that the inhibition of marrow cell DNA synthesis by SOD is specific for BFU-E and erythroid precursors (CFU-E), with other myeloid progenitors (CFU-GM) and stem cells (CFU-S) being unaffected, and IL-3 blocks the inhibitory effects of SOD on BFU-E in a dose-dependent manner. Extending earlier observations on the effects of SOD on cell proliferation, it was found that SOD was capable of inhibiting DA-1 cell proliferation supported by either IL-3 or erythropoietin (epo), but had no effect on IL-3 dependent FDCP-1 cells, nor on epo-dependent HCD-57 cells. Of several murine erythroleukemia cell lines tested, only those transformed with Friend SFFVa virus were inhibited by SOD, while those transformed with Friend SFFVp or MuLV virus were not affected. These results show that the effects of SOD are not antagonistic to particular growth factors but rather the inhibition is specific for eryrthroid cells, and cells of the proper stage can be inhibited even if they have been transformed to factor independence.     

Superoxide dismutase specifically inhibits erythroid cell DNA synthesis and proliferation.

The antioxidant enzyme superoxide dismutase (SOD) was previously shown to inhibit both the proliferation of murine erythroid DA-1 cells growing in the presence of Interleukin-3 (IL-3) and the DNA synthesis of marrow erythroid progenitor cells (BFU-E) in vitro. We show here that the inhibition of marrow cell DNA synthesis by SOD is specific for BFU-E and erythroid precursors (CFU-E), with other myeloid progenitors (CFU-GM) and stem cells (CFU-S) being unaffected, and IL-3 blocks the inhibitory effects of SOD on BFU-E in a dose-dependent manner. Extending earlier observations on the effects of SOD on cell proliferation, it was found that SOD was capable of inhibiting DA-1 cell proliferation supported by either IL-3 or erythropoietin (epo), but had no effect on IL-3 dependent FDCP-1 cells, nor on epo-dependent HCD-57 cells. Of several murine erythroleukemia cell lines tested, only those transformed with Friend SFFVa virus were inhibited by SOD, while those transformed with Friend SFFVp or MuLV virus were not affected. These results show that the effects of SOD are not antagonistic to particular growth factors but rather the inhibition is specific for erythroid cells, and cells of the proper stage can be inhibited even if they have been transformed to factor independence.     

The phosphatidylinositol 3-kinase/protein kinase B signaling pathway is activated by lipoteichoic acid and plays a role in Kupffer cell production of interleukin-6 (IL-6) and IL-10

Sepsis caused by gram-positive bacteria lacking lipopolysaccharide (LPS) has become a major and increasing cause of mortality in intensive-care units. We have recently demonstrated that the gram-positive-specific bacterial cell wall component lipoteichoic acid (LTA) stimulates the release of the proinflammatory cytokines in Kupffer cells in culture. In the present study, we have started to assess the signal transduction events by which LTA induces the production of tumor necrosis factor alpha (TNF-alpha), interleukin-6 (IL-6), and the anti-inflammatory cytokine IL-10 in rat Kupffer cells. LTA was found to trigger phosphorylation of mitogen-activated protein kinases (MAPK) (p38 MAPK and ERK 1/2) and protein kinase B (PKB). Compared to LPS, LTA was more potent in inducing PKB phosphorylation after 40 min, although we found that the cytokine responses were similar. For both bacterial molecules, blocking phosphatidylinositol 3-kinase (PI3-K; Ly294002) or Janus kinase 2 (JAK-2; AG490) particularly affected the induction of IL-6 and IL-10 release, whereas TNF-alpha levels were strongly reduced by inhibition of Src family tyrosine kinases (PP2). All three cytokines were reduced by inhibition of p38 MAPK (SB202190) or the broad-range tyrosine kinase inhibitor genistein, whereas IL-6 release was particularly blocked by inhibition of ERK 1/2 (PD98059). Divergences in the regulatory pathways controlling TNF-alpha, IL-10, and IL-6 production in Kupffer cells following LPS or LTA stimulation may create a basis for understanding how the balance between pro- and anti-inflammatory cytokines is regulated in the liver following infections by gram-positive or gram-negative bacteria.     

IL-12 and IL-10 production are differentially regulated by phosphatidylinositol 3-kinase in mast cells

The cellular mechanisms that directly regulate the production of pro- and anti-inflammatory cytokines after lipopolysaccharide (LPS) stimulation in mast cells are currently unresolved. The aim of this study was to clarify the role of phosphatidylinositol 3-kinase (PI3K) in the production of IL-12 and IL-10 in mouse bone marrow-derived mast cells (BMMCs), stimulated with Escherichia coli-derived LPS. LPS activates the PI3K signalling pathway; analysis of cytokine production following LPS stimulation of BMMCs revealed that inhibition of the PI3K pathway differentially regulated IL-10 and IL-12 syntheses. IL-12 production was enhanced, whereas IL-10 levels were suppressed. Inhibition of LPS-mediated activation of the PI3K pathway resulted in a pronounced reduction of NF-κB activity that was dependent on IκBα phosphorylation. These findings demonstrate a regulatory function for PI3K in modulating IL-10 and IL-12 production in mast cells and provide insight into how engagement of the PI3K pathway affects the induction of key immunoregulatory cytokines that control both qualitative and quantitative aspects of early inflammation.     

Amelioration of rat adjuvant arthritis by therapeutic treatment with bindarit, an inhibitor of MCP-1 and TNF-α production

OBJECTIVE: This study was designed to evaluate therapeutic effects of bindarit, an indazolic derivative able to inhibit monocyte chemoattractant protein-1 (MCP-1) production, in adjuvant induced arthritis in rats. MATERIALS AND METHODS: Arthritis was induced by Freund's complete adjuvant injection. Bindarit was given as a 0.5% medicated diet starting on day 11 after adjuvant injection. The course of arthritis was monitored by sequential paw volume measurement and by radiologic and histologic evaluations. Human osteoblast cell line Saos-2 stimulated with Interleukin-1 (IL-1) was used to assess in vitro bindarit effect on MCP-1 release. In addition, in vivo effects of bindarit on cytokine production were studied in mice injected with lipopolysaccharide (LPS). Immune function studies were performed in mice by evaluating ex vivo antibody response to ovalbumin and splenocytes proliferation to Concanavalin A (Con A). RESULTS: In adjuvant-induced arthritis in rats, bindarit possessed therapeutic activity resulting in a significant inhibition of paw inflammation. Evidence for a disease-modifying activity was also indicated by amelioration of radiologic alterations and by histological evaluation of joints. Additional evidence for beneficial effects in osseous inflammation was provided by an in vitro assay in which bindarit inhibited the release of MCP-1 from IL-1 stimulated osteoblast cells. Moreover, in a murine model of LPS-induced cytokine production bindarit reduced MCP-1 and tumor necrosis factor (TNF)-alpha increase without affecting IL-1 and IL-6 levels. Finally, the drug, given as a 0.5% medicated diet for 14 days, did not affect either anti-ovalbumin serum antibody production or splenocytes proliferative response in mice. CONCLUSIONS: Results obtained indicate that bindarit beneficial effects in experimental arthritis are correlated to MCP-1 and TNF-alpha inhibition and suggest that the control of cytokines and chemokines production can have considerable relevance as regards strategies for the treatment of chronic inflammatory diseases.     

Reduction of serum Interleukin-1-like activity after treatment with dexamethasone

The ability of steroids to modulate the appearance of Interleukin-1(IL-1) in vivo was evaluated in a model of endotoxin shock. High levels of IL-1 were found in serum from A/J mice which were sensitized with P. acnes and challenged with bacterial lipopolysaccharide (LPS). The factor appeared in the serum 2-4 hours after LPS challenge and was dependent on the period of P. acnes sensitization and the dose of LPS. Treating the mice with dexamethasone prior to LPS challenge resulted in significantly lower thymocyte proliferative activity in the serum. Three experiments demonstrated that this reduced activity reflects a decrease in IL-1. 1) The reduced activity was not due to the presence of proliferation inhibitors since mixing the serum from dexamethasone-treated mice with purified IL-1 or adding the equivalent amount of steroid directly to thymocyte cultures did not reduce the degree of proliferation. 2) When the serum was fractionated by gel filtration, the proliferative activity for both control and steroid treated sera eluted at 10-16 kilodaltons; however, the activity was nearly 50% less in the sample from steroid-treated mice. 3) In addition to thymocyte proliferative activity, IL-1 induces an increase in the serum titer of the acute phase protein known as serum amyloid A. Both serum- and gel-purified samples were able to induce the SAA, but again the samples from steroid-treated mice were much less active. We conclude that the factor produced in vivo has the properties of IL-1 and that the serum titre of the factor is reduced by dexamethasone treatment.     

The roles of interleukins in the synergistic effect of B-cell mitogens with concanavalin A on hydrocortisone-resistant thymic cell proliferation.

Investigations were made to determine the roles of interleukin (IL)-1 and IL-2 in the synergistic enhancement of DNA synthesis by concanavalin A (Con A) and bacterial lipopolysaccharide (LPS) or butanol-extracted water-soluble adjuvant (Bu-WSA) from Bacterionema matruchotii in cultures of thymic cells taken from hydrocortisone (HC)-treated C3H/HeN (LPS-responsive) and C3H/HeJ (LPS-non-responsive) mice. When the C3H/HeNCrj cells were cultured in the presence of Con A and LPS or Bu-WSA together, [3H]thymidine [( 3H]TdR) uptake of the cells was enhanced synergistically in comparison with those cultured with either one of the mitogens alone. The synergistic effect on thymic cells was dependent on Ia-positive accessory cells, since a previous treatment of the cells with anti-Iak serum and complement inhibited the response, and the inhibition could be relieved by the addition of either purified peritoneal exudate macrophages (Mø) or splenic B lymphocytes. The co-stimulation of cells with Con A and LPS or Bu-WSA also enhanced their production and release of thymic cell growth factor(s) into the culture medium. The amounts of IL-1 and IL-2 in the culture supernatants were sufficiently high to explain the activities of the growth factor(s). On the other hand, enhanced IL-2 production without significant increase in IL-1 was seen in the co-cultures of anti-Ia+ cell deprived thymic cells and purified splenic B cells prepared from C3H/HeNCrj mice in the presence of Con A and LPS or Bu-WSA, and it was seen in the cultures of C3H/HeJ thymic cells with Con A and LPS. These results suggest that the synergistic effect of Con A and LPS or Bu-WSA on the proliferative response of HC-treated thymic cells is mainly due to the enhanced production of IL-2 and its action to increase cell growth, and there are two pathways by which the enhancement of IL-2 production by Con A and LPS or Bu-WSA can occur: an IL-1-dependent pathway, or an IL-1-independent one.     

Interleukin-6 and Interleukin-6 Receptor are Expressed by Cultured Glomerular Epithelial Cells

Interleukin-6 (IL-6) has been extensively studied in mesangial cells but little is known about the expression of this cytokine and its receptor in glomerular epithelial cells (GEC). IL-6 was detected in the culture supernatants of human GEC and its production was enhanced in time and dose dependent manner by lipopolysaccharide (LPS), interleukin-1β (IL-Iβ) and tumour necrosis alpha (TNF-α). Quiescent, serum-starved GEC did not express clearly IL-6 mRNA. Stimulation of cells with LPS, TNF-α or IL-1β resulted in an increase of detectable IL-6 mRNA. Interestingly, it was found that IL-6 induced its own mRNA attesting that this cytokine was secreted in autocrine fashion by GEC. GEC expressed IL-6 receptor (IL-6R) as demonstrated directly by the existence of IL-6R mRNA detected by northern blotting. Stimulation of GEC by pro-inflammatory mediators such as LPS increased the expression of IL-6R mRNA. The soluble form of IL-6 receptor (sIL-6R) was not detectable in the culture supernatants harvested from untreated or cytokine-treated cells. We investigated further, whether IL-6 may influence growth of cultured GEC. Incubation of GEC with recombinant (r) IL-6 resulted in a dose dependent increase in ³H thymidine incorporation indicating that IL-6 acts as an autocrine growth factor for GEC. We conclude that GEC are a potent source of IL-6, the local excessive expression of IL-6 and its receptor may play a substantive role in the regulation of processes which appear critical to the initiation of progressive glomerular disease such as cell proliferation.     

Critical role of protein kinase C epsilon for lipopolysaccharide-induced IL-12 synthesis in monocyte-derived dendritic cells

In the present study we have investigated the potential involvement of protein kinase C (PKC) in the maturation of human dendritic cells (DC) by bacterial lipopolysaccharide (LPS). LPS stimulation of DC derived from human monocytes resulted in PKC phosphorylation. Inhibition of PKC activation using bisindolylmaleimide (Bis), a pan-PKC inhibitor, was associated with a dose-dependent decrease of LPS-induced IL-12 production. In contrast, up-regulation of MHC class II, CD80 and CD86 was not altered. Consistent with the diminished IL-12 synthesis, DC stimulated with LPS in presence of Bis were deficient in the induction of IFN-gamma production by allogeneic CD4+ T cells. Furthermore, we found that PKC inhibition impaired LPS-induced I kappa B-alpha degradation and subsequent nuclear factor (NF)-kappa B activation in DC. LPS resulted in the phosphorylation of conventional alpha/beta and novel epsilon PKC isoforms in DC. Inhibition of LPS-induced PKC activity using pseudosubstrate peptides specific for PKC isoforms established that PKC epsilon but not PKC alpha/beta was involved in the production of IL-12 and TNF-alpha. Overall, these data provide evidence that PKC inhibition impairs LPS signaling in DC and identify PKC epsilon as a potential target for the inhibition of Toll-like receptor-4-mediated, IL-12-dependent Th1 type responses.     

Potentiation by thiopurines and sulfhydryl-reactive agents of the inhibition by 3-deazaadenosine of mononuclear phagocytes.

Combination effects of 3-deazaadenosine (c3Ado) on antibody-dependent phagocytosis in mouse resident peritoneal cells and human peripheral blood monocytes precultured with cytotoxic thiols, azathioprine (AZA) or 6-mercaptopurine (6-MP), and thiol-reactive agents, 2-cyclohexene-1-one (2-CH) or ethacrynic acid (ETA), are described. In the mouse cell preparations, a non-inhibitory concentration of 10 microM AZA or 6-MP potentiated the inhibition by 5 and 10 microM c3Ado of phagocytosis. Higher concentrations of AZA or 6-MP (50, 100 microM) and c3Ado (40, 50 microM) were needed to achieve similar effects in human monocytes. Both 2-CH (50 microM) and ETA (25 microM) inhibited mouse cell phagocytosis and acted synergistically with c3Ado. Precultivation of mouse cells with an inhibitor of glutathione synthesis, buthionine sulfoximine (BSO, 50 microM) caused no inhibition of phagocytosis and no potentiation of the inhibition by c3Ado, although BSO potentiated the inhibition by 2-CH (50 microM). In human monocytes, non-inhibitory concentrations (10 and 25 microM) of gold sodium thiomalate (GST), AZA, and c3Ado, but not 6-MP, potentiated the inhibition by 2-CH (25-37.5 microM) of phagocytosis. Results are discussed in connection with the possible modulation by endogenous sulfhydryl-reactive metabolites of phospholipid turnover of the effects of c3Ado.     

Suppression of C3H/HeJ cell activation by lipopolysaccharide endotoxin.

Earlier studies in our laboratory showed that the lipopolysaccharide (LPS) of Salmonella typhi, which fails to activate B lymphocytes of C3H/HeJ mice, can suppress proliferation and polyclonal antibody synthesis by these cells when they are stimulated by polyclonal activators. In order to determine what stage of the cell cycle was blocked, resting B cells from C3H/HeJ spleens were activated by using different mitogens in the presence of inhibitory concentrations of LPS and analyzed by flow cytometry, using acridine orange to stain DNA and RNA. LPS was found to inhibit the progression of cells into the G1 stage of the cell cycle. Furthermore, [3H]uridine uptake studies showed that RNA synthesis is inhibited during the early phase of activation. These results indicate that inhibition by LPS of the signalling process occurs during a critical period of the cell cycle when the cells become susceptible to the inhibitory effects of LPS. To examine whether LPS acts only on B cells or whether it can suppress other immunocompetent cells from C3H/HeJ mice, studies were carried out on activated thymocytes and macrophages. LPS was found to inhibit thymocyte proliferation stimulated by concanavalin A or the combination of phorbol myristate acetate and ionomycin. Prostaglandin E2 synthesis by macrophages was also blocked by LPS. Thus, LPS is a potent inhibitor of the functioning of the major immunocompetent cells of C3H/HeJ mice.     

IL—6对小鼠（B—杂交瘤）7TD1细胞凋亡的调控作用

小鼠Ｂ－杂交瘤７ＴＤ１细胞的生长依赖于ＩＬ－６的存在。本研究对有或无ＩＬ－６条件下，对７ＴＤ１细胞的生物学行为加以分析。结果发现：１．细胞在缺乏ＩＬ－６时，生长停滞并出现细胞凋亡的典型特征；２．ＤＮＡ核酸酶抑制剂ＡＴＡ可有效防止因ＩＬ－６缺乏所致的凋亡；３．ＲＮＡ合成抑制剂ＣＨＸ能充分抑制ＩＬ－６对７ＴＤ１的生物效应，从而使细胞步入凋亡。本研究提示，ＩＬ－６通过抑制７ＴＤ１内部持续存在的细胞死亡程     

Interleukin-1 beta production in the rabbit brain during endotoxin-induced fever.

Interleukin-1 beta (IL-1 beta) production in the brain and the spleen was investigated in rabbits made febrile by intravenous (I.V.) injection of endotoxin, or human recombinant IL-1 beta (hIL-1 beta). The endotoxin used in the present study was the lipopolysaccharide (LPS) of Salmonella typhosa endotoxin. Monophasic fever was induced by I.V. injection of a low dose of LPS (0.02 micrograms kg-1) and biphasic fever by I.V. injection of a large dose of LPS (4 micrograms kg-1), a sublethal dose of LPS (40 micrograms kg-1) or hIL-1 beta (2 micrograms kg-1). In situ hybridization and immunohistochemical studies revealed that, although no IL-1 beta production was observed in the brain at 1 and 3 h after injection of a low dose of LPS (0.02 micrograms kg-1) or of hIL-1 beta (2 micrograms kg-1), IL-1 beta production was demonstrated in organum vasculosum laminae terminalis (OVLT) and some cells around the blood vessels in the parenchyma 1 h after 4 micrograms kg-1 LPS. IL-1 beta production was detected throughout the brain after 40 micrograms kg-1 LPS. Pretreatment with indomethacin, an inhibitor of prostaglandin synthesis, did not affect IL-1 beta production in the brain induced by 4 micrograms kg-1 LPS. The cell type which produces IL-1 beta in the OVLT following LPS injection was confirmed to be a macrophage by electron microscopy. The cells producing IL-1 beta in the parenchyma were determined to be microglial cells. In the spleen, each dose of LPS induced a significant increase in IL-1 beta production in polymorphonuclear cells and macrophages in the red pulp 1 h after injection. However, 2 micrograms kg-1 hIL-1 beta did not induce IL-1 beta production in the spleen. The present results show clearly that systemic administration of LPS induces IL-1 beta production in the OVLT which may be responsible for induction of the second phase of biphasic fever. The production of IL-1 beta in the OVLT was not attributable to the action of peripherally synthesized IL-1 beta or prostaglandins.     

Activated macrophage conditioned medium: identification of the soluble factors inducing cytotoxicity and the L-arginine dependent effector mechanism

Conditioned medium (CM) from cultures of cytotoxic activated macrophages causes inhibition of mitochondrial respiration, DNA synthesis, and aconitase activity in murine EMT-6 mammary adenocarcinoma cells by an L-arginine dependent effector mechanism. CM induces cytotoxicity and nitrite synthesis in EMT-6 cells in a dose dependent manner. We have identified the soluble factors in CM that induce cytotoxicity and synthesis of inorganic nitrogen oxides from L-arginine by EMT-6 cells. Using functional inhibition experiments, the activity of lipopolysaccharide (LPS), tumor necrosis factor alpha (TNF alpha), and interferon gamma (IFN gamma) in CM was investigated. The LPS inhibitor polymyxin B and TNF alpha antibody produced a modest decrease in nitrite production, while IFN gamma antibody markedly inhibited both nitrite production and cytostasis. Simultaneous treatment with polymyxin B, TNF alpha antibody, and IFN gamma antibody reduced EMT-6 cell nitrite production by 81%, and cytostasis by 74%. By Western blot, IFN gamma and TNF alpha were shown to be present in CM. When CM was subjected to hydrophobic interaction chromatography, a single peak of activity was eluted, and Western blot showed that the active fractions contained IFN gamma. Furthermore, IFN gamma antibody neutralized the activity in these chromatographic fractions. We conclude that induction of inorganic nitrogen oxide synthesis from L-arginine by the synergistic combination of IFN gamma, TNF alpha, and LPS accounts for most of the biologic activity of CM, and that IFN gamma is the major priming factor.     

Biological activity of recombinant murine interleukin-6 in interleukin-1 T cell assays

Interleukin-6 (also called B cell stimulatory factor 2, hepatocyte activating factor, interferon-β₂) has been shown to have effects on various lineages of hemopoietic cells. Some of its activities appear to overlap those of interleukin-1. In particular, recombinant murine IL-6 induced proliferation of phytohemagglutinin-activated thymocytes, an assay widely used to detect IL-1. In this report, we compared several features of IL-1 and IL-6 dependent thymocyte proliferation. The results indicate that IL-2 is the major second mediator of both IL-1 and IL-6 dependent proliferation. Finally, we tested whether IL-6 would also have activity in other T cell-based IL-1 assays using the T cell lymphoma LBRM33 1A5 and the T cell clone D10-G4.1. IL-6 had no activity in the latter two assays. These results indicate that IL-1 assays using LBRM33 1A5 and D10-G4.1 selectively detect Il-1, and are more specific assays for the detection of IL-1 in samples that may also contain IL-6.