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.     

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.     

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.     

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.     

IL-10 inhibits cytokine production by activated macrophages

IL-10 inhibits the ability of macrophage but not B cell APC to stimulate cytokine synthesis by Th1 T cell clones. In this study we have examined the direct effects of IL-10 on both macrophage cell lines and normal peritoneal macrophages. LPS (or LPS and IFN-gamma)-induced production of IL-1, IL-6, and TNF-alpha proteins was significantly inhibited by IL-10 in two macrophage cell lines. Furthermore, IL-10 appears to be a more potent inhibitor of monokine synthesis than IL-4 when added at similar concentrations. LPS or LPS- and IFN-gamma-induced expression of IL-1 alpha, IL-6, or TNF-alpha mRNA was also inhibited by IL-10 as shown by semiquantitative polymerase chain reaction or Northern blot analysis. Inhibition of LPS-induced IL-6 secretion by IL-10 was less marked in FACS-purified peritoneal macrophages than in the macrophage cell lines. However, IL-6 production by peritoneal macrophages was enhanced by addition of anti-IL-10 antibodies, implying the presence in these cultures of endogenous IL-10, which results in an intrinsic reduction of monokine synthesis after LPS activation. Consistent with this proposal, LPS-stimulated peritoneal macrophages were shown to directly produce IL-10 detectable by ELISA. Furthermore, IFN-gamma was found to enhance IL-6 production by LPS-stimulated peritoneal macrophages, and this could be explained by its suppression of IL-10 production by this same population of cells. In addition to its effects on monokine synthesis, IL-10 also induces a significant change in morphology in IFN-gamma-stimulated peritoneal macrophages. The potent action of IL-10 on the macrophage, particularly at the level of monokine production, supports an important role for this cytokine not only in the regulation of T cell responses but also in acute inflammatory responses.     

Paradoxical anti-inflammatory actions of TNF-alpha: inhibition of IL-12 and IL-23 via TNF receptor 1 in macrophages and dendritic cells

IL-12 and TNF-alpha are central proinflammatory cytokines produced by macrophages and dendritic cells. Disregulation of TNF-alpha is associated with sepsis and autoimmune diseases such as rheumatoid arthritis. However, new evidence suggests an anti-inflammatory role for TNF-alpha. TNF-alpha-treated murine macrophages produced less IL-12p70 and IL-23, after stimulation with IFN-gamma and LPS. Frequency of IL-12p40-producing macrophages correspondingly decreased as measured by intracellular cytokine staining. IL-12p40 production was also inhibited in dendritic cells. TNFR1 was established as the main receptor involved in IL-12p40 regulation, because IL-12p40 levels were not affected by TNF-alpha in TNFR1(-/-)-derived macrophages. Macrophages activated during Listeria monocytogenes infection were more susceptible to inhibition by TNF-alpha than cells from naive animals, which suggests a regulatory role for TNF-alpha in later stages of infection. This nonapoptotic anti-inflammatory regulation of IL-12 and IL-23 is an important addition to the multitude of TNF-alpha-induced responses determined by cell-specific receptor signaling.     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood. I. Comparison with isolated PBMC stimulation.

Production of interleukin 1 beta (IL-1 beta), interleukin 6 (IL-6), tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) after stimulation by lipopolysaccharide (LPS) and phytohemagglutinin (PHA) was studied in 1/10 diluted whole blood (WB) culture and in peripheral blood mononuclear cell (PBMC) culture. Cytokines IL-1 beta, TNF-alpha and IL-6 are preferentially stimulated by LPS whereas IL-2, IFN-gamma and GM-CSF are stimulated by PHA. Combination of 5 micrograms/ml PHA and 25 micrograms/ml LPS gave the most reliable production of the six cytokines studied. IL-1 beta, TNF-alpha and IL-6 represent a homogeneous group of early-produced cytokines positively correlated among themselves and with the number of monocytes in the culture (LeuM3). Furthermore, IL-1 beta was negatively correlated with the number of T8 lymphocytes. IL-2, IFN-gamma and GM-CSF represent a group of late-produced cytokines. Kinetics and production levels of IL-6 and GM-CSF are similar in WB and PBMC cultures. In contrast, production levels of TNF-alpha and IFN-gamma are higher in WB than in PBMC whereas production levels of IL-6 and IL-2 are lower in WB than in PBMC. Individual variation in responses to PHA + LPS was always higher in PBMC cultures than in WB cultures. The capacity of cytokine production in relation to the number of mononuclear cells is higher in WB, or in PBMC having the same mononuclear cell concentration as WB, than in conventional cultures of concentrated PBMC (10(6)/ml). Because it mimics the natural environment, diluted WB culture may be the most appropriate milieu in which to study cytokine production in vitro.     

Interleukin-6 and interleukin-15 are selectively regulated by lipopolysaccharide and interferon-gamma in primary pig adipocytes

3T3-L1 adipocytes express the lipopolysaccharide (LPS) receptor and respond to direct stimulation with the antigen by increasing the expression of inflammatory mediators. Activation of this receptor by its ligand in the macrophage causes the activation and translocation of nuclear factor-kappaB (NF-kappaB) to the nucleus where it regulates the expression of proinflammatory cytokines and other target genes. We investigated whether LPS could stimulate NF-kappaB translocation in primary pig adipocytes and regulate the expression and secretion of TNF-alpha and IL-6. LPS clearly induced the nuclear translocation of NF-kappaB and also upregulated (P < 0.05) the mRNA expression and secretion of IL-6 into the culture medium. An induction of TNF-alpha expression by LPS was not detected, but with extended incubation (8 h), there was a modest increase (P < 0.09) in the media concentration of this cytokine. Inhibition of either ERK1/2, PKC, or the inhibitory G protein (Gi) with U-0126, bisindolylmaleimide HCl, and pertussis toxin, respectively, blocked (P < 0.05) the increase in IL-6 expression caused by LPS. Because LPS administration in vivo increases circulating concentrations of IFN-gamma, and because this cytokine also regulates multiple immune modulators in the adipocyte, we also determined whether IFN-gamma regulates cytokine expression in primary adipocytes. Although the expression of IL-6 and TNF-alpha was unresponsive to IFN-gamma, the expression of IL-15 was markedly upregulated (P < 0.01). Furthermore, the induction of IL-15 expression by IFN-gamma was blocked by inhibition of PKC. These data indicate that NF-kappaB is responsive to LPS in the adipocyte and also identify key mediators of LPS-induced IL-6 expression. In addition, we provide novel evidence that IFN-gamma targets the adipocyte to induce IL-15 expression, thus indicating a possible role for the adipocyte in the regulation of T-cell function and muscle metabolism during the innate immune response.     

Lipopolysaccharide induces rapid production of IL-10 by monocytes in the presence of apoptotic neutrophils

There is growing evidence that apoptotic neutrophils have an active role to play in the regulation and resolution of inflammation following phagocytosis by macrophages and dendritic cells. However, their influence on activated blood monocytes, freshly recruited to sites of inflammation, has not been defined. In this work, we examined the effect of apoptotic neutrophils on cytokine production by LPS-activated monocytes. Monocytes stimulated with LPS in the presence of apoptotic neutrophils for 18 h elicited an immunosuppressive cytokine response, with enhanced IL-10 and TGF-beta production and only minimal TNF-alpha and IL-1beta cytokine production. Time-kinetic studies demonstrated that IL-10 production was markedly accelerated in the presence of apoptotic neutrophils, whereas there was a sustained reduction in the production of TNF-alpha and IL-1beta. This suppression of proinflammatory production was not reversible by depletion of IL-10 or TGF-beta or by addition of exogenous IFN-gamma. It was demonstrated, using Transwell experiments, that monocyte-apoptotic cell contact was required for induction of the immunosuppressive monocyte response. The response of monocytes contrasted with that of human monocyte-derived macrophages in which there was a reduction in IL-10 production. We conclude from these data that interaction between activated monocytes and apoptotic neutrophils creates a unique response, which changes an activated monocyte from being a promoter of the inflammatory cascade into a cell primed to deactivate itself and other cells.     

Modulation of cytokine release from mononuclear cells by prostacyclin, IL-4 and IL-13

In a previous study, we reported that cicaprost, a stable prostacyclin analogue can inhibit the release of granulocyte-macrophage colony-stimulating factor (GM-CSF) from activated human peripheral mononuclear blood cells (PBMCs). Since interleukin (IL-4) and IL-13 have been shown to inhibit the release of cytokines from PBMCs we tested the hypothesis that prostacyclin in combination with IL-4 or IL-13 can act synergistically to modulate the release of IL-10, generally associated with anti-inflammatory properties, and the pro-inflammatory cytokine tumour necrosis factor alpha (TNF-alpha). For this purpose, PBMCs were isolated over Ficoll, stimulated with lipopolysaccharide (LPS) and incubated in the presence of cicaprost, IL-4 or IL-13. There was a significant reduction in TNF-alpha as well as IL-10 secretion from LPS-stimulated PBMCs following incubation with IL-4 or IL-13. In contrast, cicaprost reduced the secretion of TNF-alpha but led to a slight enhancement of IL-10 release from PBMCs. When LPS-activated PBMCs were incubated in the presence of cicaprost and IL-4 or IL-13 there was a selective, synergistic inhibition of the TNF-alpha release which was not observed for IL-10. Thus, our data suggest that prostacyclin can synergize with cytokines to selectively inhibit the release of pro-inflammatory cytokines from PBMCs.     

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.     

Critical paracrine interactions between TNF-alpha and IL-10 regulate lipopolysaccharide-stimulated human choriodecidual cytokine and prostaglandin E2 production

Increased production of PGs by gestational membranes is believed to be a principal initiator of term and preterm labor. Intrauterine infection is associated with an inflammatory response in the choriodecidua characterized by elevated production of cytokines and PGs. The precise physiological significance of enhanced choriodecidual cytokine production in the mechanism of preterm labor remains uncertain. These studies were undertaken to dissect the roles and regulation of endogenous cytokines in regulating PG production by human choriodecidua. We used LPS treatment of human choriodecidual explants as our model system. In choriodecidual explant cultures, LPS (5 microg/ml) induced a rapid increase in TNF-alpha production, peaking at 4 h. In contrast, IL-10, IL-1beta, and PGE2 production rates peaked 8, 12, and 24 h, respectively, after LPS stimulation. Immunoneutralization studies indicated that TNF-alpha was a primary regulator of IL-1beta, IL-10, and PGE2 production, while IL-1beta stimulated only PGE2 production. Neutralization of endogenous IL-10 resulted in increased TNF-alpha and PGE2 production. IL-10 treatment markedly decreased TNF-alpha and IL-1beta production, but had no effect on PGE2 production. Taken together, these results demonstrate that the effects of LPS on choriodecidual cytokine and PG production are modulated by both positive and negative feedback loops. In the setting of an infection of the intrauterine, TNF-alpha may be a potential target for treatment intervention; IL-10 could be one such therapeutic.     

Direct stimulation of cytokines (IL-1 beta, TNF-alpha, IL-6, IL-2, IFN-gamma and GM-CSF) in whole blood: II. Application to rheumatoid arthritis and osteoarthritis.

Rheumatoid arthritis (RA) is an immune disease in which the pathological immune reaction is thought to be initiated by the presentation of an (auto) antigen or superantigen by MHC class II positive cells to CD4 T cells. These successive immunological events can be studied by the cytokines produced at the different stages. Cytokine secretion by stimulated cells in autologous diluted whole blood has allowed the study of the immune profile characteristic of rheumatoid arthritis. The pattern of RA patient whole blood cells cultured in autologous blood is characterized by hyperactivity of the mononuclear cells with high secretion of IL-1 beta, TNF-alpha and IL-6 and low production of IFN-gamma, in comparison with the normal (N) and osteoarthrosis (OA) populations. The IL-2 secretion pattern is unique, arising from production followed by consumption. This production-consumption turnover is the most elevated in the RA group. The T cells are indeed activated in rheumatoid arthritis but regulatory events suppress some of their functions. A correlation was found between the inflammatory proteins and mediators of cellular immunity and macrophagic function: IL-1 beta and the sedimentation rate; IL-6 and fibrinogen; TNF-alpha and the number of blood monocytes. The secretion of OA-stimulated whole blood cells was similar to RA for two monokines (overproduction of TNF-alpha and IL-6) and different for IL-1 beta, not different from normal in OA. Stimulated whole blood cell cytokine secretion profile from RA and OA groups, was the same as previously observed in synovial fluid.(ABSTRACT TRUNCATED AT 250 WORDS)