Differential effects of neuropeptides on cytokine production by mouse helper T cell subsets

DNA damage caused by tamoxifen and its derivatives was examined by estimating the conversion of supercoiled pUC18 plasmid DNA to linear form by means of agarose gel electrophoresis. N-Desmethyltamoxifen induced DNA cleavage and its effect was enhanced by the addition of reducing agents such as dithiothreitol, NADPH and 2-mercaptoethanol. 4-Hydroxytamoxifen itself had little effect, but the cleavage was slightly enhanced by the addition of reducing agents. DNA damage was higher with alpha-hydroxytoremifene than with alpha-hydroxytamoxifen, which had a prominent effect only at high concentration. The cleavage by alpha-hydroxy derivatives were not enhanced by reducing agents. No damage was induced by tamoxifen, toremifene, 3-hydroxytamoxifen or N-desmethyltoremifene. The DNA cleavage by N-desmethyltamoxifen was inhibited by the addition of EDTA, mannitol, sodium azide, methionine, catalase and superoxide dismutase. The formation of 8-hydroxy-2'-deoxyguanosine was also examined with calf thymus DNA in vitro. A slight increase of its level was found with 4-hydroxytamoxifen in the presence of dithiothreitol and also with N-desmethyltamoxifen in the presence of NADPH, but alpha-hydroxytoremifene and alpha-hydroxytamoxifen were ineffective. These experimental data suggest that among metabolites of tamoxifen, N-desmethyltamoxifen and probably also 4-hydroxytamoxifen cause oxidative DNA damage in which redox cycling is involved. The DNA damage by alpha-hydroxytoremifene appears to involve a different mechanism from that by N-desmethyltamoxifen. Tamoxifen and toremifene are possibly metabolized to the forms contributing to DNA damage.     

Effects of dental amalgam and heavy metal cations on cytokine production by peripheral blood mononuclear cells in vitro

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is known to produce a differential toxicity in the nigrostriatal and mesolimbic dopaminergic pathways with the nigrostriatal pathway being more vulnerable. We, therefore, investigated whether oxidative stress and the antioxidant system play a role in this phenomenon. Balb/c mice were treated with either saline or MPTP (30 mg/kg/d) for 7 d, and were sacrificed on the next day. Results revealed that MPTP increased lipid peroxidation in the striatum (ST) and decreased glutathione concentration in the substantia nigra (SN) without markedly affecting these measures in the nucleus accumbens (NAc) and ventral tegmental area (VTA). Further, MPTP produced approximately twofold increases in both manganese superoxide dismutase (MnSOD) and copper-zinc superoxide dismutase (CuZnSOD) activities in the VTA while it only increased MnSOD activity in the SN. Both catalase and glutathione peroxidase (GPx) activities were not markedly altered by MPTP in both systems. However, the basal levels of catalase and GPx activities were higher in the VTA and NAc than in the SN and ST. These results together suggest that a lesser degree of oxidative damage and a more inducible CuZnSOD activity observed in the mesolimbic dopaminergic pathway may partially explain the differential toxicity MPTP produced in these two dopaminergic systems.     

Effects of chromium extract on cytokine release by mononuclear cells

We have evaluated the effects of chromium extract on the release by peripheral blood mononuclear cells (PBMCs) of cytokines favouring bone resorption. Furthermore, we have evaluated whether the chromium effects could be correlated with the activation and proliferation of PBMCs. Cell cultures were maintained in serum-free medium (AIM-V), in order to avoid the interference of exogenous growth factors. Increasing concentrations of chromium extract, ranging between 3 and 100%, were added to culture medium. Cytokine release (IL-1beta, TNFalpha, IL-6, GM-CSF and IFNgamma) was assessed on both PBMCs cultured with AIM-V only (unstimulated PBMC) and PBMCs cultured with AIM-V plus phytohaemagglutinina (PHA-stimulated PBMC). The activation and proliferation of PBMCs were evaluated by assessing DNA synthesis and soluble IL-2 receptor release, in order to determine whether an IL-2-dependent immune response can be induced by chromium. Our results show that in unstimulated PBMCs chromium ions slightly increased the release of pro-inflammatory cytokines, such as TNFalpha and IL-6, even though the increase is not significant. On the contrary, the different concentrations of chromium extract significantly inhibited the response to PHA stimulation, as shown by the decrease in IL-6 and sIL-2r release, and by the influence on cell viability and DNA synthesis. Both these effects are undesirable and support hypotheses on the biological effects of chromium. The continuous release of chromium from the implant could induce in PBMCs the release of bone-resorbing cytokines, which in the long term could be responsible for irreversible tissue damage. Moreover, chromium seems to inhibit the IL-2-dependent response of PBMCs, so that they are not able to trigger an efficient cell-mediated immune response.     

Effects of free radical scavengers on cytokine actions on islet cells

We investigated the effect of free radical scavengers on the actions of cytokines on islet cells. Interferon-gamma and tumor necrosis factor-alpha reduced the nicotinamide adenine dinucleotide content of mouse islet cells; the combination of interferon-gamma (4 x 10(5) U/l) and tumor necrosis factor-alpha (4 x 10(5) U/l) caused nicotinamide adenine dinucleotide reduction by approximately 40%. Dimethyl urea and dimethyl sulfoxide prevented the decrease, whereas superoxide dismutase, catalase, and mannitol were not effective. Dimethyl urea and dimethyl sulfoxide protected islet cells from the synergistic cytotoxic action of interferon-gamma and tumor necrosis factor-alpha. Major histocompatibility complex class II antigen induction by interferon-gamma and tumor necrosis factor-alpha was also inhibited by dimethyl urea and dimethyl sulfoxide, but not by superoxide dismutase, catalase and mannitol. Since superoxide dismutase of a membrane-penetrable form attenuated the class II antigen induction, the inefficiency of superoxide dismutase, catalase and mannitol may be attributable to their inability to penetrate islet cells. These results suggest that the intracellular generation of free oxygen radicals is involved in islet cell cytotoxicity and class II molecule expression by interferon-gamma and tumor necrosis factor-alpha, and that nicotinamide adenine dinucleotide reduction may be associated with islet cell dysfunction caused by the cytokines.     

Effect of cytokine-specific antisense oligonucleotides on the immunoglobulin production by rat spleen cells in vitro

We examined in this study the regulation of immunoglobulin (IgM, IgG, IgE) production by spleen cells from N brasiliensis infected rats following addition of antisense oligodeoxynucleotides (ODNs). The ODNs were selected near the AUG initiation codon of mRNA specific for interleukin 4 (IL-4) or interleukin 2 (IL-2). Results show that addition of antisense to IL-4 inhibited IgE production, while the production of IgG and IgM increased. The use of sense IL-4 sequence did not affect immunoglobulin production. In contrast, the use of antisense IL-2 ODN induced an enhancement of IgE as well as of IgM and IgG responses. Both the Ig secretion in culture supernatants and the number of Ig secreting cells, as detected by an Elispot assay, were influenced by the presence of antisense IL-4 ODNs. These results clearly show the involvement of IL-2 and IL-4 in the regulation of isotype selection during antibody synthesis and that IL-2 and IL-4 do operate differently on IgE production. They also argue that antisense strategy represents a useful tool for the antibody regulation.     

Analysis of cytokine production by inflammatory mouse macrophages at the single-cell level: selective impairment of IL-12 induction in Leishmania-infected cells

Intracellular staining for cytokines and parasites, combined with two-color flow cytometric analyses, were used to examine the frequencies of IL-12-, TNF-alpha- and IL-6-producing macrophages in response to Leishmania major infection and/or activation with IFN-gamma/lipopolysaccharide (LPS). Inflammatory macrophages were obtained from nonimmune granulomas, initiated by the injection of polyacrylamide microbeads (Bio-gel P-100) into subcutaneous pouches of different mouse strains. Infection of inflammatory macrophages in vitro using metacyclic promastigotes produced identical effects on cytokine responses regardless of whether cells from genetically resistant or susceptible mouse strains were used: IL-12 was not produced in response to infection itself, virtually every infected cell lost its ability to produce IL-12 in response to IFN-gamma/LPS, and the IL-6 response was partially inhibited, while the TNF-alpha response of infected cells was unimpaired. Low-multiplicity infection of inflammatory macrophages in vivo using either metacyclic promastigotes or tissue amastigotes also resulted in the complete and selective inhibition of IL-12 responses in infected cells. These data establish the physiologic relevance of prior observations regarding the selective impairment of IL-12 induction pathways in infected macrophages, and suggest a mechanisms for the delayed onset of cell-mediated control mechanisms that is typical of even self-limiting forms of leishmanial disease.     

Effects of methotrexate on differentiation of monocytes and production of cytokine inhibitors by monocytes

OBJECTIVE: To examine the potential of methotrexate (MTX) to act as a differentiation-stimulating factor for monocytes, which could explain the antiinflammatory properties of this agent in the treatment of rheumatoid arthritis (RA). METHODS: Fluorescence-activated cell sorter analysis was used to measure the changes in antigen expression (CD11b/c, CD16, CD64, CD14, CD68, and CD95) in response to MTX, 1,25-OH-cholecalciferol (1,25-OH-CCF), and granulocyte-macrophage colony-stimulating factor in the human monoblastic leukemia cell line U937, bone marrow mononuclear cells (BMMC), and peripheral blood mononuclear cells (PBMC). Release of interleukin-1beta (IL-1beta), IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor a, and soluble tumor necrosis factor receptors (sTNFR) p55 and p75 during the differentiation in vitro was assessed by immunoassay in the culture supernatants. RESULTS: MTX alone and in combination with 1,25-OH-CCF markedly stimulated the differentiation of the monocytic U937 cells and simultaneously increased Fas-antigen expression. Differentiation was associated with enhanced IL-1Ra and sTNFR p75 release from U937 cells. MTX had fewer effects on phenotypic differentiation of human BMMC and PBMC, but did stimulate IL-1Ra release and inhibit IL-1beta synthesis in BMMC. CONCLUSION: MTX acts as a strong differentiation factor for immature and undifferentiated monocytic cells. Differentiation in vitro is associated with an increase in natural cytokine inhibitor release and a simultaneous down-regulation of IL-1beta. These findings may explain the marked clinical antiinflammatory effects of MTX when used in the treatment of RA.     

CD40 ligand stimulates proinflammatory cytokine production by human endothelial cells

Functional expression of CD40 has recently been described on the surface of HUVEC, and activation of these cells with CD40 ligand (CD40-L) leads to increased adhesion molecule expression. Here, we analyzed the effect of CD40 triggering on cytokine production by HUVEC. CD40-L-transfected fibroblasts, in contrast to their untransfected counterparts, as well as a soluble recombinant human CD40-L/murine CD8alpha chimeric molecule were able to importantly increase (by a mean of fourfold) the production of leukemia inhibitory factor (LIF) by HUVEC. CD40-L displayed an additive effect with IL-4, IL-1alpha, and TNF-alpha on the enhancement of LIF secretion. Cyclosporin A did not affect LIF synthesis by resting or CD40-L-activated HUVEC, whereas dexamethasone diminished the basal level of LIF production and abrogated the CD40-L effect. The secretions of two other proinflammatory cytokines, granulocyte-macrophage CSF and IL-6, were also increased in the presence of CD40-L. However, CD40-L neither affected HUVEC proliferation nor rescued them from IFN-gamma- and TNF-alpha induced apoptosis. Together, these results indicate that endothelial cell activation by CD40-L may play an important role not only in leukocyte recruitment through enhancement of adhesion molecule expression, but also in the maintenance of an inflammatory loop through the increase in proinflammatory cytokine secretion.     

Tumor necrosis factor production in the perfused mouse liver and its pharmacological modulation by methylxanthines

The liver contains the largest pool of cytokine-producing macrophages in the body and may therefore play an important role in the development and outcome of systemic inflammatory response syndromes. Therefore, we investigated the tumor necrosis factor-alpha (TNF) releasing capacity of the in situ perfused mouse liver and its modulation by methylxanthines, i.e., by a class of well-established inflammatory cytokine-suppressing drugs. We have shown that pretreatment of mice with either lipopolysaccharide or TNF elicited a dose-dependent TNF release into the perfusate which was inhibited by in vivo pretreatment of mice with pentoxifylline or A-802715 [1-(5-hydroxy-5-methyl)hexyl-3-methyl-7-propylxanthin]. Infusion of these methylxanthines into livers from mice pretreated with lipopolysaccharide or TNF also inhibited TNF release in an immediate and reversible way even after TNF production had been initiated. The inhibitory effect of methylxanthines was prevented by pretreatment of mice with the adenylate cyclase inhibitor dideoxyadenosine, suggesting upregulation of the cyclic adenosine monophosphate system as a possible mechanism of action of these drugs. Our findings demonstrate that the liver is a potent cytokine producer and identify it as one of the target organs of methylxanthines or other phosphodiesterase inhibitors in murine models of shock and inflammatory liver failure.     

Shiga toxin-1 regulation of cytokine production by human proximal tubule cells

BACKGROUND: Interleukin-1 (IL-1), interleukin-6 (IL-6), and tumor necrosis factor (TNF) levels are elevated in kidneys of patients with post-diarrheal hemolytic uremic syndrome (D+HUS) and may contribute to renal dysfunction. The renal cellular sources of these inflammatory cytokines in D+HUS are largely unknown, however, the proximal tubule has emerged as a potentially important candidate. Since Shiga toxin-1 (Stx-1) has been implicated in the genesis of D+HUS, we examined the effect of Stx-1 on cytokine production by human proximal tubule cells. METHODS: Stx-1 cytotoxicity, protein synthesis inhibition, and effect on IL-1, IL-6, and TNF protein release and mRNA levels were determined. The effect of another protein synthesis inhibitor, cycloheximide (CHX), on these parameters was also evaluated. RESULTS: Stx-1 greatly increased TNF release and mRNA levels while CHX, at concentrations that produced similar inhibition of protein synthesis, had no effect on TNF production. In contrast, Stx-1 and CHX caused comparable elevations in IL-1 release and mRNA accumulation. Stx-1 and CHX also stimulated IL-6 mRNA accumulation, but only at concentrations that either were cytotoxic or substantially blocked protein synthesis. Finally, lipopolysaccharide, which is likely to be elevated in the circulation of patients with D+HUS, had no effect alone, but synergized with Stx-1 to increase IL-1 production. CONCLUSIONS: These results indicate that Stx-1 stimulates proximal tubule inflammatory cytokine production and that this effect is due partially to nonspecific induction of mRNA levels as well as activation of Stx-1-specific mechanisms.     

Correlation of antibiotic-induced endotoxin release and cytokine production in Escherichia coli-inoculated mouse whole blood ex vivo

Escherichia coli were incubated in mouse whole blood ex vivo supplemented with beta-lactam antibiotics that possessed preferential affinities for penicillin-binding proteins (PBPs). After 4 h, viable bacteria were undetectable in the presence of any of the 3 antibiotics tested, whereas significant increases in colony-forming units were detected in samples not treated with antibiotics. Differential levels of endotoxin in platelet-rich plasma were detected using the limulus amebocyte lysate assay, according to differential antibiotic affinities for the various PBPs. Levels of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) in antibiotic-treated cultures after 8 h of incubation correlated well with the levels of endotoxin at 4 h (r = .96, P < .0001 for TNF-alpha; r = .91, P = .0002 for IL-6). These data indicate that differential affinities of beta-lactam antibiotics for PBPs affect both endotoxin and cytokine responses ex vivo in mouse blood and correlate with in vivo protective efficacy of these antibiotics in gram-negative experimental models.     

Analysis of the in vitro cytokine production by liver-infiltrating T cells of patients with autoimmune hepatitis

The pathogenic mechanisms underlying the development of autoimmune hepatitis (AIH) are still unclear. Since AIH is associated with the presence of various autoantibodies and certain HLA subtypes, it is likely that T and B cells play a major role in this disease. In this study we have determined the functional capacities of in vivo preactivated liver-infiltrating T cells (LTC) from patients with AIH. As controls we used LTC from patients with non-autoimmune hepatitis (non-AIH). Our results show that preactivated LTC from patients with AIH predominantly (190/255 clones) reside in the CD4+ population, whereas LTC in non-AIH are dominated by the CD8+ phenotype (148/254 clones). In view of this finding we have investigated the cytokine secretion patterns of 102 randomly chosen CD4+ T cell clones from six patients with AIH. As controls we have used 58 CD4+ LTC from 11 patients with non-AIH. All clones were stimulated by lectin and irradiated accessory cells and subsequent cytokine production was evaluated. LTC from patients with AIH have a lower interferon-gamma (IFN-gamma)/IL-4 ratio compared with LTC from non-AIH. Although clones from some patients with AIH produced very high amounts of IL-4 in vitro, this was not a constant finding. These results show that in vivo preactivated LTC from patients with AIH are mostly CD4+ T cells that produce more IL-4 than IFN-gamma. In contrast, LTC from patients with non-AIH are dominated by CD8+ and CD4+ T cells that produce significantly less IL-4 than IFN-gamma. Thus, liver-infiltrating T cells from patients with AIH and non-AIH belong to different functional T cell subsets. This may have implications for the regulation of humoral and cellular immune responses in inflammatory liver disease.     

Effects of bacterial DNA on cytokine production by (NZB/NZW)F1 mice

Microbial DNA has multiple immune effects including the capacity to induce polyclonal B cell activation and cytokine production in normal mice. We recently described the accelerated induction of anti-DNA Abs in NZB/NZW mice immunized with Escherichia coli (EC) dsDNA; paradoxically these mice developed less renal disease than unimmunized mice or mice immunized with calf thymus DNA. We postulated that alterations in cytokine production induced by bacterial DNA may play a key role in renal protection. To determine the effect of bacterial DNA on cytokine production in NZB/NZW mice, we measured the serum cytokine levels, cell culture supernatant cytokine levels, and number of cytokine-producing splenocytes in NZB/NZW mice injected with EC DNA, calf thymus DNA, or an immune active oligonucleotide. There was a 10- to 25-fold increase in the number of cells secreting IFN-gamma compared with IL-4 in mice immunized with EC DNA. IL-12-secreting cells were also increased by bacterial DNA immunization. In parallel with the increase in IFN-gamma secreting cells, there was a significant rise in serum IFN-gamma levels in mice receiving EC DNA. These results indicate that EC DNA modulates systemic cytokine levels in NZB/NZW mice, selectively increasing IL-12 and IFN-gamma while decreasing IL-4 production. The cytokine response of NZB/NZW mice to bacterial DNA may be of significance in disease pathogenesis and relevant to the treatment of lupus-like disease.