Inflammatory cytokine production in interferon-gamma-primed mice, challenged with lipopolysaccharide. Inhibition by SK&F 86002 and interleukin-1 beta-converting enzyme inhibitor

Mice challenged with lipopolysaccharide (LPS) produce variable serum levels of pro-inflammatory cytokines, and particularly low levels of interleukin-1 beta (IL-1 beta). Interferon-gamma (IFN-gamma) has been shown to be an important mediator of bacteria-induced hypersensitivity to LPS in mice. In the present study, we show that mice pretreated with IFN-gamma exhibit an enhanced capacity to produce serum IL-1 beta, IL-1 alpha, tumour necrosis factor (TNF-alpha) as well as IL-6 in response to LPS. Priming with intraperitoneal (i.p.) injection of 15 mg rat recombinant IFN-gamma, 18 hours prior to the i.p. LPS (300 mg) challenge resulted in a 4-fold increase in the LPS-stimulated release of IL-1 beta and a 2- to 7-fold increase in the release of IL-1 alpha, TNF-alpha, as well as IL-6 into the serum. LPS induced a concentration-dependent increase in the release of IL-1 beta in isolated peritoneal macrophages from IFN-gamma-primed mice whereas macrophages from unprimed mice released minute amounts of IL-1 beta. In addition, nigericin markedly enhanced the release of IL-1 beta in unprimed mice but not in macrophages from IFN-gamma primed mice. The cytokine synthesis inhibitor SK&F 86002, administered per os (100 mg/kg), 1 hour prior to LPS challenge, strongly inhibited the rise in serum levels of the four cytokines. Furthermore, treatment with the IL-1 beta converting enzyme (ICE) specific reversible inhibitor YVAD-CHO resulted in a sharp dose- and time-dependent inhibition of IL-1 beta secretion in the serum, whereas the other cytokines were not affected. In conclusion, IFN-gamma priming strongly potentiates the release of proinflammatory cytokines in the serum of mice as compared to LPS stimulation alone, and provides therefore a useful way to test the in vivo potency and selectivity of cytokine synthesis inhibitors.     

Cytokine-mediated modulation of leptin and adiponectin secretion during in vitro adipogenesis: evidence that tumor necrosis factor-alpha- and interleukin-1beta-treated human preadipocytes are potent leptin producers

Over the last decade, compelling evidence has been presented that cytokines affect adipocyte tissue formation and function. In this study we explored the effect of pro-inflammatory (i.e. interleukin (IL)-1beta, IL-6, interferon (IFN)-gamma, and tumor necrosis factor (TNF)-alpha) versus anti-inflammatory cytokines (i.e. IL-4, IL-10, and transforming growth factor (TGF)-beta1) on leptin and adiponectin secretion during in vitro human adipogenesis. Confirmative to previous reports, conversion of precursor preadipocytes into mature adipocytes was completely inhibited upon exposure to TNF-alpha, IL-1beta, IFN-gamma, or TGF-beta1. Hence, all these anti-adipogenic cytokines prevented release of adipocyte-specific adiponectin. IFN-gamma also strongly reduced leptin production (> or =85%). However, TNF-alpha, IL-1beta, and TGF-beta1 stimulated leptin production from preadipocytes in the absence of mature adipocytes (20.6+/-5.4 ng/ml, 100.8+/-18.2 ng/ml, and 5.4+/-0.4 ng/ml, respectively, compared to 6.6+/-0.8 ng/ml in control adipocyte cultures on day 21; n=4). IL-4, IL-6 and IL-10 did not, or only slightly, affect adipocyte differentiation and their hormonal secretion. In conclusion, adiponectin and leptin are both synthesized by adipocytes, whereas leptin is also produced by preadipocytes upon TNF-alpha or IL-1beta stimulation. These data suggest that preadipocytes could contribute more to total circulating leptin levels than has been previously considered, especially in diseased conditions were these pro-inflammatory factors play a prominent role.     

Control of apolipoprotein E secretion in the human hepatoma cell line KYN-2

Even though it is known that apolipoprotein E (apoE) is deeply involved in major age-related disorders such as atherosclerosis or Alzheimer's disease (AD), the control of cell-specific apoE expression is still poorly understood. We compared the apoE secretion as previously described in astrocytic cell17 to hepatic cell apoE secretion. We used the human hepatoma cell line KYN-2 to better delineate the characteristics of apoE secretion and to validate it with respect to the classical human hepatoma cell line HepG2. Interleukin-1beta (IL-1beta) and interferon-gamma (IFN-gamma) significantly inhibited, while IL-2, IL-6 and tumour necrosis factor-alpha (TNF-alpha) were inactive on apoE secretion by KYN-2 as well as HepG2 cells. Cholesterol and 25-OH cholesterol had no effect, while forskolin exerted a significant inhibitory effect, on apoE secretion in KYN-2 cells. Our results suggest that the KYN-2 cell line represents an appropriate cell model, and in any case an alternative model to the HepG2 cell line, to study the control of apoE secretion. The response of KYN-2 cells to both cytokines and cholesterol differs from that found in astrocytoma cells, suggesting that blood variations of apoE concentrations in AD may not reflect the dysregulations taking place in the brain.     

Apoprotein E isoform-dependent expression and secretion of pro-inflammatory cytokines TNF-alpha and IL-6 in macrophages

The anti-atherogenic properties of human apoprotein E-associated lipoproteins have been partially attributed to its anti-inflammatory properties. We studied if endogenously expressed apoprotein E (apoE) elicits isoform-dependent effects on pro-inflammatory cytokine expression and secretion. Mouse J774A.1 peritoneal macrophages without native expression of apoE were used to establish cell lines with stable expression of the three human apoE isoforms, apoE2, apoE3 and apoE4. In the presence of lipopolysaccharide (LPS), expression and secretion of TNF-alpha and IL-6 in cells expressing different apoE isoforms were determined by RT-PCR, immunoblotting and ELISA assays. ApoE3-expressing cells have significantly lower expression and secretion levels of the two cytokines as compared to cells with apoE2 and apoE4 expression. Such observations were accompanied with the lowest ERK1/2 activity in apoE3-expressing cells. Further study shows that the apoE isoform-dependent variations of TNF-alpha and IL-6 expression/secretion in macrophages are diminished in the presence of ERK1/2 inhibitor U0126. In conclusion, apoE elicits isoform-dependent effects on macrophage TNF-alpha and IL-6 expression as well as secretion. The ERK1/2 signaling pathways are involved in mediating such apoE isoform-dependent effects.     

Corticosteroids and interferons inhibit cytokine-induced production of IL-8 by human endothelial cells

IL-8, secreted by endothelial cells at the site of inflammation, participates in recruitment and transmigration of leukocytes. IL-8 may also have pathophysiological consequences in inflammatory and immunological disorders. We have investigated the effect of interferons (IFNs) and glucocorticosteroids (GCs) on cytokine induced secretion and production of IL-8 by human umbilical endothelial cells (HUVEC). There was a low spontaneous secretion of IL-8 by unstimulated HUVEC which increased after 6 or 24 h of stimulation with the pro-inflammatory cytokines TNF-alpha or IL-1beta. IFN-gamma as well as the GCs, Dexamethasone and Budesonide, inhibited TNF-alpha induced IL-8 secretion in a dose-dependent manner. IFNs may have a general modulating effect, since IFN-alpha also inhibited the TNF-alpha-induced IL-8 secretion. There was a slight, but significant, increase in the content of intracellular IL-8 in stimulated HUVEC. However, there was no difference between stimulation with IL-1beta or TNF-alpha alone or in combination with IFNs or GCs, whereas inhibition of IL-8 secretion with monensin increased IL-8 content suggesting that IFNs and GCs inhibit synthesis rather than secretion of IL-8. In conclusion, IFNs or GCs may be useful for inhibiting IL-8 production by endothelial cells and could thus be used for therapeutic modulation of the inflammatory response.     

Outer membrane vesicles from Neisseria meningitidis: effects on cytokine production in human whole blood

The Norwegian group B meningococcal outer membrane vesicle (OMV) vaccine consists of outer membrane proteins (OMPs) as main antigens with significant amounts of lipopolysaccharide (LPS; 5-9% relative to protein). We have studied the ability of this OMV vaccine preparation to induce secretion of pro-inflammatory cytokines, tumour necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), interleukin 6 (IL-6), interleukin 8 (IL-8) and anti-inflammatory cytokines, interleukin 4 (IL-4), interleukin 10 (IL-10) and interleukin 13 (IL-13) in a human whole blood model. Plasma levels of TNF-alpha, IL-1beta, IL-6 and IL-8 were massively increased; mean peak levels of TNF-alpha 44 696+/-7764, IL-1beta 38 043+/-5411, IL-6 10 057+/-1619 and IL-8 30 449+/-5397 pg/ml were obtained with an OMV-LPS concentration of 1 microg/ml; corresponding levels in control plasmas were below the detection limit of the assay. Mean maximal level of IL-10 (2540+/-144 pg/ml) was obtained at OMV-LPS concentration of 10 microg/ml, after 24 h; while the level in control plasma was below detection limit. OMV-LPS did not induce release of IL-4 and IL-13 in doses from 0.001-10 microg/ml. The present results show that OMVs from meningococci have potent pro-inflammatory properties and are likely to contribute to the observed local and systemic inflammatory effects.     

Comparative effects of cytokines and cytokine combinations on complement component C3 secretion by HepG2 cells

The mechanisms that control complement protein synthesis are incompletely understood. Recent evidence suggests that cytokines are involved in the regulation of hepatic synthesis of circulating complement components. Therefore, we compared the effects of human recombinant IL-1alpha, IL-1beta, IL-6, IFN-gamma, and TNF-alpha individually or in combination, on HepG2 secretion of complement component C3, the major opsonic protein of the complement system. HepG2 cells were incubated with each cytokine alone and with various combinations of the cytokines. At 24, 48, 72, and 96 h of incubation, the C3 and albumin secreted by the HepG2 cells were quantified by a sandwich ELISA. IL-1alpha and IFN-gamma significantly enhanced C3 secretion by the cells (P<0.02 vs. control cells). IL-1beta when combined with either IL-6 or IFN-gamma also increased C3 secretion (P<0.03 vs. control cells). The stimulatory effect on HepG2 cells by the IL-1beta/IL-6 combination was synergistic. With the exception of IL-1alpha, which increased albumin secretion, HepG2 secretion of albumin was not affected by incubation with individual cytokines or the cytokine combinations. Therefore, IL-1alpha, IFN-gamma, and the combination of IL-1beta with IL-6 or IFN-gamma specifically enhanced C3 secretion by HepG2 cells. The greatest magnitude of C3 secretion was induced by the combination of IL-1beta and IL-6.     

Recombinant rat IL-1beta and IL-6 synergistically enhance C3 mRNA levels and complement component C3 secretion by H-35 rat hepatoma cells

Hepatic synthesis of complement component C3 is regulated in part by inflammatory cytokines. Rat models are frequently employed to investigate pathogenic roles of complement and cytokines. However, cytokines obtained from species other than the rat were used in previous studies of cytokine regulation of C3 synthesis in rat hepatocytes or hepatoma cells. It is not known whether these prior reports predict hepatocellular responses evoked by rat cytokines. Therefore, H-35 rat hepatoma cells were employed to measure the effect of recombinant rat IL-1beta, IL-6, IFN-gamma, and TNF-alpha on C3 protein secretion and C3 mRNA levels quantified by ELISA and quantitative RT-PCR. Compared to untreated control cells, H-35 cells treated with IL-1beta, IL-6, and IFN-gamma increased C3 secretion approximately 10-, 4-, and 2-fold, respectively. TNF-alpha was toxic, precluding further analysis. IL-1beta and IL-6 demonstrated synergy with respect to the quantity and rate of increase of C3 mRNA measured and the magnitude of C3 protein secretion. Previous reports using non-rat cytokines did not consistently predict H-35 responses to rat cytokines. Consequently, we recommend the use of rat cytokines in rat models that include analysis of cytokine-mediated events.     

Tumor necrosis factor-alpha production by astrocytes. Induction by lipopolysaccharide, IFN-gamma, and IL-1 beta

Astrocytes have the capacity to secrete or respond to a variety of cytokines including IL-1, IL-6, IL-3, and TNF-alpha. In this study, we have examined the capacity of astrocytes to secrete TNF-alpha in response to a variety of biologic stimuli, particularly cytokines such as IL-1 and IFN-gamma, which are known to be present in the central nervous system during neurologic diseases associated with inflammation. Rat astrocytes do not constitutively produce TNF-alpha, but have the ability to secrete TNF-alpha in response to LPS, and can be primed by IFN-gamma to respond to a suboptimal dose of LPS. IFN-gamma and IL-1 beta alone do not induce TNF-alpha production, however, the combined treatment of IFN-gamma and IL-1 beta results in a striking synergistic effect on astrocyte TNF-alpha production. Astrocyte TNF-alpha protein production induced by a combined treatment of either IFN-gamma/LPS or IFN-gamma/IL-1 beta occurs in a dose- and time-dependent manner, and appears to require a "priming signal" initiated by IFN-gamma, which then renders the astrocyte responsive to either a suboptimal dose of LPS or IL-1 beta. Astrocyte TNF-alpha production by IFN-gamma/LPS stimulation can be inhibited by the addition of anti-rat IFN-gamma antibody, whereas IFN-gamma/IL-1-induced TNF-alpha production is inhibited by antibody to either IFN-gamma or IL-1 beta. Polyclonal antisera reactive with mouse macrophage-derived TNF-alpha neutralized the cytotoxicity of IFN-gamma/LPS and IFN-gamma/IL-1 beta-induced astrocyte TNF-alpha, demonstrating similarities between these two sources of TNF-alpha. We propose that astrocyte-produced TNF-alpha may have a pivotal role in augmenting intracerebral immune responses and inflammatory demyelination due to its diverse functional effects on glial cells such as oligodendrocytes and astrocytes themselves.     

Toll-like receptor (TLR) 2-9 agonists-induced cytokines and chemokines: I. Comparison with T cell receptor-induced responses

The cells of innate and adaptive immunity, although activated by different ligands, engage in cross talk to ensure a successful immune outcome. To better understand this interaction, we examined the demographic picture of individual TLR (TLRs 2-9) -driven profiles of eleven cytokines (IFN-alpha/beta, IFN-gamma, IL-12p40/IL-12p70, IL-4, 1L-13, TNF-alpha, IL-1beta, IL-2, IL-10) and four chemokines (MCP-1, MIP1beta, IL-8, and RANTES), and compared them with direct T-cell receptor triggered responses in an assay platform using human PBMCs. We find that T-cell activation by a combination of anti-CD3/anti-CD28/PHA induced a dominant IL-2, IL-13, and Type-II interferon (IFN-gamma) response without major IL-12 and little Type-I interferon (IFN-alphabeta) release. In contrast, TLR7 and TLR9 agonists induced high levels of Type-I interferons. The highest IFN-gamma levels were displayed by TLR8 and TLR7/8 agonists, which also induced the highest levels of pro-inflammatory cytokines IL-12, TNF-alpha, and IL-1beta. Amongst endosomal TLRs, TLR7 displayed a unique profile producing weak IL-12, IFN-gamma, TNF-alpha, IL-1beta, and IL-8. TLR7 and TLR9 resembled each other in their cytokine profile but differed in MIP-1beta and MCP1 chemokine profiles. Gram positive (TLR2, TLR2/6) and gram negative (TLR4) pathogen-derived TLR agonists displayed significant similarities in profile, but not in potency. TLR5 and TLR2/6 agonists paralleled TLR2 and TLR4 in generating pro-inflammatory chemokines MCP-1, MIP-1beta, RANTES, and IL-8 but yielded weak TNF-alpha and IL-1 responses. Taken together, the data show that diverse TLR agonists, despite their operation through common pathways induce distinct cytokine/chemokine profiles that in turn have little or no overlap with TCR-mediated response.     

IgE-dependent cytokine production by human peripheral blood mononuclear phagocytes.

These studies demonstrate the IgE-dependent production of IL-1 beta and TNF-alpha by circulating blood monocytes. IL-1 beta production was demonstrated biologically as the stimulation of proliferation of the cloned IL-1-dependent murine T cell line D10.G4.1 in the presence of a submitogenic concentration of PHA. In a representative experiment, 3H-thymidine uptake increased from 57826 cpm in the presence of supernatants obtained from unstimulated cells to 200774 cpm with supernatants from monocytes stimulated by IgE/alpha IgE immune complexes. By ELISA, IgE complexes increased IL-1 beta production from 0.54 +/- 0.06 ng (per 10(6) monocytes) to 2.60 +/- 0.62 ng (p less than 0.01; mean of eight experiments) and TNF-alpha production from 0.17 +/- 0.10 ng to 3.00 +/- 0.54 ng (p less than 0.01; mean of four experiments). No IL-1 alpha secretion was observed. RNA hybridization analysis demonstrated that IL-1 beta production represented de novo synthesis of the cytokine. Stimulated RNA production was observed after a minimal 1/2-h incubation and was maximal at 2 h. The IgE-dependent secretion of these pro-inflammatory cytokines by mononuclear phagocytic cells may contribute to the inflammation characteristic of allergic responses.     

The effect of fish oil supplementation on cytokine production in children

The ex vivo production of inflammatory cytokines during fish oil supplementation (n-3 polyunsaturated fatty acids, n-3 PUFA) is a matter of considerable controversy. Studies on human subjects have generally reported decreased lymphocyte proliferation and decreased production of IL-2, interferon-gamma, IL-1beta, IL-6 and TNF-alpha, but other studies showed no effect or even increased production. There are no published reports on ex vivo cytokine production in children on long-term, n-3 PUFA supplementation. The current double-blind study explored cytokine production by peripheral blood mononuclear cells (PBMCs), with and without lipopolysaccharide (LPS) stimulation in children on 12 weeks' supplementation with 300 mg/day of n-3 PUFA. Twenty-one children (aged 8-12 years) were randomized to receive 1 g canola oil (control) or 300 mg n-3 PUFA + 700 mg canola oil in a chocolate spread. Blood was then drawn and PBMCs were separated and cultured for 24 h in a culture medium with or without 10 microg/mL LPS for 5 x 10(6) PBMCs. The pro-inflammatory cytokines, IL-1beta, TNF-alpha and IL-6, and the anti-inflammatory cytokines, IL-10 and IL-1RA, were evaluated by ELISA. The levels of all the cytokines were higher in non-stimulated and LPS-stimulated cultures, from n-3 PUFA-treated subjects as compared to controls. There was no difference in the IL-1beta/IL-1RA ratio between the two groups, with and without LPS stimulation. Nevertheless, the ratio tended to be lower in the treated subjects on both occasions. In conclusion, our results indicate an increased production of both pro-inflammatory and anti-inflammatory cytokines, with and without LPS stimulation, in children on 12 weeks' n-3 PUFA supplementation.     

Adenosine - cyclic AMP pathways and cytokine expression.

Adenosine and cAMP are potent modulators of immune-triggered cytokine production. Their effects overlap with regard to the inhibition of the pro-inflammatory cytokines TNF-alpha, IFN-gamma, IL-12, and the stimulation of production of the major anti-inflammatory cytokine IL-10. They may tentatively be considered to be upregulators of the production of Th2 cytokines (IL-10, IL-6), but downregulators of the production of Th1 cytokines (IL-2 and IFN-gamma). Cytokines produced in common by Th0, Th1 and Th2 cells are affected as well, although the low quantity and heterogeneity of the contemporary experimental data do not allow unambiguous conclusions to be drawn. Nevertheless, IL-3, IL-4, MIP-1alpha/beta and GM-CSF have usually been found to be inhibited, IL-5 stimulated, while IL-1 remains largely unaffected by adenosine or cAMP. These effects, and in particular the inhibition of TNF-alpha and stimulation of IL-10 expression, might be of therapeutic value in a variety of pathophysiological conditions.     

Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages. Comparison of activating cytokines and evidence for independent production

The capacity of 12 cytokines to induce NO2- or H2O2 release from murine peritoneal macrophages was tested by using resident macrophages, or macrophages elicited with periodate, casein, or thioglycollate broth. Elevated H2O2 release in response to PMA was observed in resident macrophages after a 48-h incubation with IFN-gamma, TNF-alpha, TNF-beta, or CSF-GM. Of these, only IFN-gamma induced substantial NO2- secretion during the culture period. The cytokines inactive in both assays under the conditions tested were IL-1 beta, IL-2, IL-3, IL-4, IFN-alpha, IFN-beta, CSF-M, and transforming growth factor-beta 1. Incubation of macrophages with IFN-gamma for 48 h in the presence of LPS inhibited H2O2 production but augmented NO2- release, whereas incubation in the presence of the arginine analog NG-monomethylarginine inhibited NO2- release but not H2O2 production. Although neither TNF-alpha nor TNF-beta induced NO2- synthesis on its own, addition of either cytokine together with IFN-gamma increased macrophage NO2- production up to six-fold over that in macrophages treated with IFN-gamma alone. Moreover, IFN-alpha or IFN-beta in combination with LPS could also induce NO2- production in macrophages, as was previously reported for IFN-gamma plus LPS. These data suggest that: 1) tested as a sole agent, IFN-gamma was the only one of the 12 cytokines capable of inducing both NO2- and H2O2 release; 2) the pathways leading to secretion of H2O2 and NO2- are independent; 3) either IFN-gamma and TNF-alpha/beta or IFN-alpha/beta/gamma and LPS can interact synergistically to induce NO2- release.     

Proinflammatory cytokines in bovine colostrum potentiate the mitogenic response of peripheral blood mononuclear cells from newborn calves through IL-2 and CD25 expression

Abstract: Bovine colostrum contains high concentrations of cytokines, and colostral cytokines are considered to be an important factor in stimulation of maturation of the immune system in newborns. In this study, 5 proinflammatory cytokines (IL-1beta, IL-6, TNF-alpha, IFN-gamma and IL-1 receptor antagonist, IL-1ra) present in colostrum were tested for their potential to enhance mitogenic response and to elicit expression of IL-2 mRNA and CD25 in peripheral blood mononuclear cells (PBMC) from newborn calves before being fed colostrum. PBMC were pretreated with each recombinant bovine cytokine for 2 hr before stimulation with concanavalin A (ConA). Pretreatment of PBMC from newborn calves with IL-1beta, TNF-alpha or IFN-gamma significantly enhanced the ConA response, whereas IL-1ra inhibited the response. The degree of enhancement or inhibition of mitogenic response by these cytokines was more pronounced in PBMC from newborn calves than in those from adult cows. Although IL-2 mRNA expression in ConA-stimulated PBMC from newborn calves was weaker than that in those from adult cows of ConA-stimulated controls, the expression levels became comparable after pretreatment with IL-1beta, TNF-alpha or IFN-gamma. The CD25 expression in PBMC from newborn calves was also enhanced by pretreatment with IL-1beta, TNF-beta and IFN-gamma. These results suggest that pretreatment of neonatal PBMC with IL-1beta, TNF-alpha or IFN-gamma promotes mitogenic response to ConA through up-regulating the production of IL-2 and the expression of the mature IL-2 receptor.