Stimulation of interleukin-6 release by interleukin-1beta from isolated human adipocytes

The secretion of interleukin-6 (IL-6) is modulated by immune, hormonal and metabolic stimuli in a cell-specific manner. We investigated the effect of cytokines, TNFalpha and IL-1beta, and insulin on IL-6 release from human adipocytes and peripheral blood cells (PBC). Adipocytes released IL-6 constitutively (after 5 h: 5.64 [1.61-15.30]pg ml(-1), after 10 h: 15.95 [2.34-45.59]pg ml(-1), p = 0.007), while PBC secretion did not change significantly over this period. LPS stimulated IL-6 secretion in PBC after 5 h but was without effect on adipocytes. TNFalpha and insulin induced IL-6 production from PBC, but had no effect on adipocytes. IL-1beta, however, induced a substantial increase in IL-6 release in adipocytes and PBC (all p < 0.05). Adipose tissue production of IL-1beta was assessed in vivo by measuring arterio-venous differences across the subcutaneous abdominal adipose bed. Net release of IL-1beta was not observed, suggesting that under basal conditions there is no detectable release of this cytokine into the circulation from this depot. In conclusion (1) PBC demonstrate regulated IL-6 release, while the adipocyte release has a large constitutive component; (2) immune modulators, such as LPS, TNFalpha and IL-1beta, all induce PBC IL-6 release, but only IL-1beta stimulates adipocyte release. Though IL-1beta is not an endocrine signal from adipose tissue, it is an autocrine/paracrine stimulator of IL-6 release from human adipocytes.     

Human dendritic cells discriminate between viable and killed Toxoplasma gondii tachyzoites: dendritic cell activation after infection with viable parasites results in CD28 and CD40 ligand signaling that controls IL-12-dependent and -independent T cell production of IFN-gamma

We studied how the interaction between human dendritic cells (DC) and Toxoplasma gondii influences the generation of cell-mediated immunity against the parasite. We demonstrate that viable, but not killed, tachyzoites of T. gondii altered the phenotype of immature DC. DC infected with viable parasites up-regulated the expression of CD40, CD80, CD86, and HLA-DR and down-regulated expression of CD115. These changes are indicative of DC activation induced by T. gondii. Viable and killed tachyzoites had contrasting effects on cytokine production. DC infected with viable T. gondii rather than DC that phagocytosed killed parasites induced secretion of high amounts of IFN-gamma by T cells from T. gondii-seronegative donors. IFN-gamma production in response to DC infected with viable parasites required CD28 and CD40 ligand (CD40L) signaling. In addition, this IFN-gamma response was dependent in part on IL-12 secretion. Production of IL-12 p70 occurred after interaction between T cells and DC infected with viable T. gondii, but not after incubation of T cells with DC plus killed tachyzoites. IL-12 synthesis was inhibited by blockade of CD40L signaling. IL-12-independent IFN-gamma production required CD80/CD86-CD28 interaction and, to a lesser extent, CD40-CD40L signaling. Taken together, T. gondii-induced activation of human DC is associated with T cell production of IFN-gamma through CD40-CD40L-dependent release of IL-12 and through CD80/CD86-CD28 and CD40-CD40L signaling that mediate IFN-gamma secretion even in the absence of bioactive IL-12.     

Substance P and calcitonin gene-related peptide increase IL-1 beta, IL-6 and TNF alpha secretion from human peripheral blood mononuclear cells.

We found that substance P (SP) and calcitonin gene-related peptide (CGRP) (0.3-1 microM) increased, in a concentration-dependent manner, the basal secretion of interleukin-1 beta (IL-1 beta), interleukin-6 (IL-6), and tumor necrosis factor alpha (TNF alpha) from cultured lymphocyte-enriched mononuclear cells isolated from human peripheral blood. SP and CGRP (0.1 microM) synergistically increased basal TNF alpha secretion. Dynorphin A((1-17)) (0.1-1 microM) did not modify basal cytokine secretion. Lipopolysaccharide (10 ng/ml)-induced cytokine secretion and [(3)H]thymidine uptake were not altered by any neuropeptide (at 0.1 microM). Thus, SP and CGRP stimulate the production of pro-inflammatory cytokines from lymphocytes only at high concentrations, similar to those reached during tissue damage.     

Suppressed cytokine and immunoglobulin secretions by murine splenic lymphocytes infected in vitro with Toxoplasma gondii tachyzoites

Mechanisms of host immunosuppression after infection with Toxoplasma gondii are unclear. This study was performed to observe cytokine and immunoglobulin secretions by murine splenic lymphocytes infected in vitro with live, nonreplicating (irradiated) RH tachyzoites on stimulation with concanavalin A (Con A) or lipopolysaccharide (LPS). For lymphocyte cultivation, 3 groups were prepared: coculture with live nonirradiated tachyzoites separated by a transwell (group T), live irradiated tachyzoites without a transwell (group R), and no tachyzoites (group C). Compared with group T, groups R and C, on stimulation with Con A, revealed significantly (P < 0.05) lower levels of interleukin-2 (IL-2) and IFN-gamma, but not IL-10. The levels of IgG1, IgG2a, IgG2b, IgG3, IgA, and IgM were also significantly (P < 0.05) lower in groups R and C than in group T after stimulation with LPS. The results suggest that intracellular infection of murine splenic lymphocytes with T. gondii tachyzoites could impair their capacity to produce cytokine and immunoglobulin secretions.     

Migration and maturation of human dendritic cells infected with Toxoplasma gondii depend on parasite strain type

Migration and maturation of human dendritic cells derived from CD34+ progenitor cells (DC) infected by Toxoplasma gondii were studied in an in vitro model. We demonstrated that infection with virulent type I strains RH and ENT or type II low virulent strains PRU and CAL induced DC migration towards MIP-3beta. However, type II strains induced a higher percentage of migrating cells than that induced by type I strains or positive controls (chemical allergen or lipopolysaccharides). Type II strains produced soluble factors responsible of the high migration whereas heat killed tachyzoites did not induced a migration higher than positive controls. We also demonstrated that infection by virulent strains and not by type II stains or heat killed tachyzoites triggers DC maturation. A soluble factor released by type II strains was responsible of the absence of DC maturation. Taken together, these results demonstrated that the interference of T. gondii in the behaviour of DC functions is related to the strain types and can be supported by secretion of soluble factors by the parasite.     

Cytokine and intracellular signaling regulation of tissue factor expression in astrocytes

There is evidence that inflammatory cytokines such as IL-1beta, TNFalpha, and IL-6 are involved in the pathogenesis of cerebrovascular disorders including stroke. One action of cytokines that contributes to diseases in peripheral tissues is upregulation of the procoagulant receptor tissue factor (TF). In the CNS, astrocytes are the primary cells that express TF; although little is known about how TF is regulated in these cells. Experiments were performed to evaluate the effect of cytokine treatment on TF activity in primary cultures of murine cortical astrocytes and in the human astrocytoma cell line (CCF). IL-1beta treatment induced a 2.5-fold increase in TF activity in the primary astrocytes and a 3-fold induction in the astrocytoma cells. TNFalpha treatment induced a 2.5-fold increase in TF activity in both the primary astrocytes and astrocytoma cells. IL-6 upregulated TF activity 2-fold in primary astrocytes, however, it had no effect on TF activity in the astrocytoma cells. The signaling pathways regulating TF expression in these cells were examined by using staurosporine, a broad spectrum inhibitor of serine-threonine protein kinases, and by examining the effects of intermediates in the sphingomyelin signaling pathway. Staurosporine inhibited IL-1beta-induced TF activity in the primary astrocytes but did not effect IL-1beta- or TNFalpha-induced TF activity in the astrocytoma cells. TF activity in the astrocytoma cells was upregulated 1.5-fold over constitutive levels by a ceramide analogue or the enzyme sphingomyelinase, however the ceramide analogue had no effect on TF activity in the primary astrocytes. These results suggest inflammatory cytokines can upregulate TF activity in astrocytes and the astrocytoma CCF cell line although the two cell types appear to utilize different signaling pathways to mediate TF expression. Further studies will be important to more completely define the signaling regulation of TF in astrocytes since alterations in brain TF levels may play a key role in CNS pathophysiology.     

Glucocorticoid-regulated adipose tissue secretion of PAI-1, but not IL-6, TNFalpha or leptin in vivo.

OBJECTIVE: Glucocorticoids are well-known regulators of adipose tissue metabolism and endocrine function. The aim of this study was to examine glucocorticoid effect on plasminogen activator inhibitor 1 (PAI-1), tumour necrosis factor alpha (TNFalpha), leptin and interleukin-6 (IL-6) adipose tissue secretion. MATERIAL AND METHODS: Twelve healthy postmenopausal women with mean BMI of 28.9 kg/m(2) (+/- 0.8 SEM) received 25 mg prednisolone daily for 7 days. Before and after glucocorticoid treatment adipose tissue secretion of PAI-1, leptin, IL-6 and TNFalpha were measured, and adipocyte PAI-1 mRNA as well as anthropometrical and bio-chemical data were obtained. RESULTS: Anthropometric measurements remained unaffected. Analyses of venous blood-samples showed a borderline increase of insulin levels (p = 0.062). PAI-1 secretion from adipose tissue increased (1.9 +/- 0.2 vs. 3.5 +/- 0.5 ng/g triglycerides, p = 0.012), but PAI-1 mRNA levels did not (0.19 +/- 0.02 vs. 0.21 +/- 0.04 arbitrary units after normalised to beta-actin, p = 0.51). There were no apparent differences in IL-6, TNFalpha or leptin secretion after glucocorticoid exposure. CONCLUSION: This study shows an increased secretion of PAI-1, but not IL-6, TNFalpha or leptin, from abdominal adipose tissue after in vivo glucocorticoid treatment, which may be a finding of pathophysiological importance given the well-known effect of glucocorticoid excess on metabolic aberrations and cardiovascular morbidity.     

The effect of tumor necrosis factor-alpha (TNF-alpha), interleukin (IL)-1 beta and IL-6 on chorioamnion secretion of prostaglandins (PG)F 2 alpha and E2 in pigs

The aim of the present study was to determine the effect of tumor necrosis factor-alpha (TNF-alpha), interleukin-1 beta (IL-1 beta) and interleukin-6 (IL-6) on prostaglandin (PG)F(2 alpha) and PGE(2) secretion as well as cyclooxygenase-2 (COX-2) protein expression in chorioamnion collected on days 25, 30 and 40 of pregnancy in pigs. Fetal membrane slices were incubated for 16 h with TNF-alpha, IL-1 beta, IL-6 (1 or 10 ng/ml of medium) or two combinations of the three cytokines (1 or 10 ng/ml of each cytokine per combination). We demonstrated the stimulatory effect of TNF-alpha, IL-1 beta and/or IL-6 on PGF(2 alpha) and PGE(2) secretion by the porcine fetal membranes. The medium content of these PGs depended on the cytokine type, treatment dose and day of pregnancy. Cytokine stimulation of PGE(2) was more pronounced than that of PGF(2 alpha). In addition, an increase in PGF(2 alpha) and/or PGE(2) secretion was usually associated with an augmentation of COX-2 protein expression. Our results support the notion concerning the possible role of cytokines in modulating production of PGs by fetal membranes during the first trimester of gestation.     

Interleukin-1 alpha and tumor necrosis factor-alpha (TNF alpha) inhibit growth and induce TNF messenger RNA in MCF-7 human breast cancer cells.

We studied the effects of interleukin-1 alpha (IL-1) and tumor necrosis factor-alpha (TNF), alone and in combination, on MCF-7 breast cancer cells to determine whether these cytokines alter cell growth, TNF gene expression, and TNF secretion. We found that IL-1 alone and TNF alone inhibited cell growth in a dose-dependent manner. Each cytokine arrested growth in the G0/G1 phase of the cell cycle, with maximum growth inhibition at 1000 U/ml (P less than 0.05) and 100 U/ml (P less than 0.01), respectively. However, the combination of these two cytokines did not result in greater growth inhibition or a greater percentage of cells arrested in the G0/G1 phase of the cell cycle compared with each cytokine alone. We examined the effect of exogenous IL-1 and TNF on TNF gene expression by Northern blot analysis. In the absence of any cytokine, these cells do not express TNF mRNA. Exposure to IL-1 (1000 U/ml) induced TNF mRNA at 3 h; however, mRNA levels diminished thereafter to barely detectable levels by 24 h. Exposure to TNF (1000 U/ml) also induced TNF mRNA at 3 h, but in contrast to IL-1, the level of enhanced expression persisted at these levels through 72 h of exposure. Secretion of TNF by these cells is induced by exogenous TNF, but not by IL-1. IL-1 and TNF in combination do not produce greater inhibition of growth, greater amounts of TNF mRNA at 3 h, or greater secretion of TNF than that produced by TNF alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyvalent 23 epitope polysaccharide pneumonia vaccine induced effective protection through strain-adapted effector mechanisms as demonstrated by the different cytokine responses in mice challenged with two different strains of Streptococcus pneumoniae

We used a Balb/c mouse model of pneumococcal pneumonia to investigate the protection mechanisms induced by immunization with a polyvalent 23 epitope polysaccharide pneumonia vaccine. Groups of mice were injected x 4 times s.c. within one month, with this vaccine preparation. Mice were subsequently challenged at day 45, with a lethal, intratracheal inoculum of two strains of Streptococcus pneumoniae - either a highly virulent and strongly immunogenic serotype 3 strain (P4241), or a less virulent and weakly immunogenic serotype 19F strain (P15986). The intratracheal S. pneumoniae challenge-induced lethality, antibody response, bacterial clearance, and cytokine secretions were monitored to analyze the strain-adapted effector mechanisms. Pulmonary levels of TNFalpha, IL-6, IL-1 beta, MIP-1 alpha, KC, MCP-1/JE and MIP-2 cytokines were determined up to 48 hours post-infection. Survival rates were 82% and 100% among vaccinated animals challenged at day 45 with P4241, and P1598 mice respectively, and 0% in non-vaccinated mice (p<0.001). Survival was associated with a rapid bacterial clearance from blood and lungs, which similar for the two strains. Immunization induced a serotype-specific antibody response. Kinetics of the cytokine profile in the lung following intratracheal inoculation with the 4241 strain was different in animals vaccinated 45 days previously, compared to na?ve, control mice. Generally speaking the bacterial-induced inflammatory cytokine response induced with the 4241 strain was much weaker in vaccinated animals than in control mice. The only cytokines showing a greater increase in vaccinated mice compare to control animals were IL-1 beta, KC and MCP-1. Production of TNFalpha and IL-6 was lower in vaccinated animals than in controls. At variance with the previous bacteria strain-induced cytokine profile, infection with the P15986 strain induced a strong inflammatory response, with a substantial increase in all the cytokine tested, which was similar in vaccinated and in na?ve, control animals, except for MIP-1 alpha, which was the only mediator significantly more produced by vaccinated animals than by na?ve, control mice following P15986 infection. The distinct cytokine profiles, which were observed in this study depending upon the two strains of S. pneumoniae used for challenge, demonstrated that protection against each strain was obtained through a different defence strategy.     

Role of IL-6 in the induction of hepatic metallothionein in mice after partial hepatectomy

Metallothioneins (MT) are induced upon partial hepatectomy (PH), possibly mediated by various cytokines. In the present study, we studied cytokine-dependent MT synthesis in partially hepatectomized IL-6 gene knock-out (GKO) mice in the remaining lobe of the liver. We focused on IL-6, TNFalpha and IL-1beta, the major cytokines thought to be involved in MT synthesis. The IL-6 GKO mice and B6J129Sv (wild-type control) mice were subjected to 70% PH or laparotomy. We found that MT was significantly decreased in IL-6 GKO mice, although PH induced hepatic MT in both strains of mice. Laparotomy induced MT in the liver of wild-type mice but not in IL-6 GKO mice. Pretreatment of IL-6 GKO mice with rIL-6 (5 microg/mouse) restored hepatic MT synthesis. Serum IL-6 level in wild-type mice was maximal at 6 h after surgery and decreased thereafter. Serum IL-1beta was the same in both strains of mice. Serum TNFalpha basal level in IL-6 GKO mice was higher than in wild-type mice. PH caused an increase in serum TNFalpha level in both strains of mice, and it was two times higher in IL-6 GKO mice than in wild-type mice at 18 h after surgery. We conclude that IL-6 plays a predominant role in hepatic MT synthesis after PH, but that IL-6 GKO mice still reserve the capacity to synthesize MT by an as yet unidentified mechanism.     

A role for protein kinase C activity in interleukin-1 (IL-1) induction of IL-2 gene expression but not in IL-1 signal transduction.

Interleukin-1 (IL-1) is known to synergize with phorbol esters in the induction of interleukin-2 (IL-2) expression in T-lymphoid leukemia cells and proliferation of mouse thymocytes. We used a plasmid construct containing the bacterial gene for chloramphenicol acetyltransferase under the control of the human IL-2 promoter to study the nature of this synergism in the murine thymoma cell line EL4. Although IL-1 induction of the IL-2 promoter in these cells required costimulus with phorbol myristate acetate, the signal induced by IL-1 was qualitatively different. We provide evidence to support the hypothesis that the phorbol ester signal is mediated by protein kinase C, and we show that the IL-1 signal is not. That IL-1 and phorbol myristate acetate represent different stimuli was shown by their response to protein kinase C inhibitors, capacity to synergize with increased intracellular free calcium, and requirement for protein synthesis. In addition we show that pretreatment with IL-1 can prime EL4 cells to subsequent activation by concentrations of phorbol esters not normally sufficient to induce IL-2 expression. Pretreated cells remained primed for at least 40 h after removal of the IL-1. Neither phorbol myristate acetate nor a calcium ionophore was capable of preactivating EL4 cells.     

Hypoxia increases production of interleukin-1 and tumor necrosis factor by human mononuclear cells

Exposure to hypoxia (PO2 = 9 +/- 1 torr) increased human peripheral blood mononuclear cell production and secretion of interleukin-1 (IL-1)alpha, IL-1 beta, and tumor necrosis factor (TNF) percent of control = 190% for IL-1 alpha, p = 0.014; 219% for IL-1 beta, p = 0.014; and 243% for TNF, p = 0.037) following treatment with endotoxin (1 ng/ml). Hypoxia potentiated the increased production of these inflammatory cytokines at subthreshold levels of endotoxin with potentiation increasing at lower O2 concentrations. Hypoxia also increased cytokine production induced by the tumor promoter phorbol myristate acetate, suggesting a generalized biologic response. We conclude that hypoxia increases IL-1 and TNF production and speculate that this mechanism aggravates a variety of pathologic conditions involving endotoxin such as adult respiratory distress syndrome (ARDS), multiple organ failure, and septic shock.     

Inhibition of insulin secretion from rat islets of Langerhans by interleukin-6. An effect distinct from that of interleukin-1.

Glucose-induced insulin secretion from islets cultured in the presence of interleukin-6 (IL-6) for 12-24 h was inhibited to a similar extent as when islets were treated with interleukin-1 beta (IL-1 beta). However, unlike IL-1 beta, IL-6 did not potentiate insulin secretion during an acute (30 min) exposure of islets to the cytokine, nor did it inhibit DNA synthesis during a 24 h culture period. A 12 h pretreatment of islets with tumour necrosis factor-alpha (TNF-alpha) combined with IL-1 beta potentiated the inhibitory effect of IL-1 beta on secretion, such that 20 mM-glucose-induced insulin secretion was abolished. No synergistic inhibition of secretion was observed with TNF-alpha and IL-6. However, IL-1 beta and IL-6 were found to inhibit insulin secretion in an additive manner. These results suggest that IL-6 inhibits insulin secretion in a manner distinct from that of IL-1 beta, and that IL-6 is unlikely to mediate the inhibitory effects of IL-1 beta or TNF-alpha on rat islets of Langerhans.     

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.