Plasma interleukin-6, tumour necrosis factor alpha and blood cytokine production in type 2 diabetes

Type 2 diabetes is associated with increased circulating concentrations of markers of the acute-phase response and interleukin-6 (IL-6). An augmented acute-phase response may be a mechanism which explains many of the clinical and biochemical features of type 2 diabetes and its complications. We sought to confirm that circulating concentrations of the cytokine acute-phase mediators IL-6 and tumour necrosis factor alpha [TNFalpha] are elevated in type 2 diabetes, and investigated blood as a source of cytokines in type 2 diabetes. Blood samples from 20 type 2 diabetic and 17 age-matched healthy subjects were incubated in vitro for 24 hr with and without lipopolysaccharide (LPS) stimulation and secreted cytokines measured. Plasma IL-6 and TNFalpha were significantly increased in type 2 diabetes compared to normal subjects. However, basal production of IL-6 and TNFalpha in cultured diabetic blood was markedly depressed in comparison with non-diabetic samples. IL-6 and TNFalpha production was increased in blood in response to LPS, reaching similar levels in diabetic and non-diabetic subjects, though IL-6 was slightly but significantly higher in controls. We conclude that circulating levels of IL-6 and TNFalpha are increased in type 2 diabetes but there is downregulation of basal cytokine production in blood cells in type 2 diabetes. Blood has the capacity to produce cytokines in diabetes which contribute to the augmented acute-phase response, but the main source of the increased plasma IL-6 and TNFalpha concentrations may be from non-circulating cells.     

Magnesium decreases inflammatory cytokine production: a novel innate immunomodulatory mechanism

MgSO(4) exposure before preterm birth is neuroprotective, reducing the risk of cerebral palsy and major motor dysfunction. Neonatal inflammatory cytokine levels correlate with neurologic outcome, leading us to assess the effect of MgSO(4) on cytokine production in humans. We found reduced maternal TNF-α and IL-6 production following in vivo MgSO(4) treatment. Short-term exposure to a clinically effective MgSO(4) concentration in vitro substantially reduced the frequency of neonatal monocytes producing TNF-α and IL-6 under constitutive and TLR-stimulated conditions, decreasing cytokine gene and protein expression, without influencing cell viability or phagocytic function. In summary, MgSO(4) reduced cytokine production in intrapartum women, term and preterm neonates, demonstrating effectiveness in those at risk for inflammation-associated adverse perinatal outcomes. By probing the mechanism of decreased cytokine production, we found that the immunomodulatory effect was mediated by magnesium and not the sulfate moiety, and it was reversible. Cellular magnesium content increased rapidly upon MgSO(4) exposure, and reduced cytokine production occurred following stimulation with different TLR ligands as well as when magnesium was added after TLR stimulation, strongly suggesting that magnesium acts intracellularly. Magnesium increased basal I?Bα levels, and upon TLR stimulation was associated with reduced NF-κB activation and nuclear localization. These findings establish a new paradigm for innate immunoregulation, whereby magnesium plays a critical regulatory role in NF-κB activation, cytokine production, and disease pathogenesis.     

Arg-vasopressin increases proliferation of human osteoblast-like cells and decreases production of interleukin-6 and macrophage colony-stimulating factor

Patients with arginine-vasopressin (AVP) deficiency have been reported to have a decreased bone mass. The mechanism behind this is not known. In this study, the effects of AVP on primary human osteoblast-like (hOB) cells and SaOS-2 cells were investigated. Cell proliferation was measured by [3H]thymidine incorporation or a commercially available kit (EZ4U), and protein synthesis by [3H]proline incorporation. In addition, the production of interleukin-6 (IL-6) and macrophage colony-stimulating factor (M-CSF) in hOB cells was determined. AVP at 10-100 pmol/l increased cell proliferation in hOB and SaOS-2 cells (p < 0.05). Protein synthesis increased in SaOS-2 cells incubated with 10-100 pmol/l AVP (p < 0.01). When hOB and SaOS-2 cells were incubated with AVP together with a vasopressin receptor-1 (V1)-antagonist ([beta-Mercapto-beta,beta-cyclopenta-methylenepropionyl1,O-Me-Tyr2,Arg8]-vasopressin) or a protein kinase C (PKC)-inhibitor (chelerythrine) the increase in cell proliferation in response to AVP was abolished. The production of IL-6 and M-CSF was decreased in hOB-cells incubated with 10 pmol/l AVP (p < 0.01). In addition, by RT-PCR, we found evidence for expression of mRNA for the vasopressin 1a (V1a)-receptor in hOB cells. In conclusion, AVP stimulated proliferation of hOB- and SaOS-2 cells. We suggest that the effect was mediated through the V1-receptor. Additionally, AVP decreased production of IL-6 and M-CSF from the hOB cells. Moreover, the V1a-receptor seems to be expressed in hOB cells.     

Erythropoietin as an antiapoptotic, tissue-protective cytokine

Erythropoietin (EPO) increases the number of circulating erythrocytes primarily by preventing apoptosis of erythroid progenitors. In addition to this proerythroid action, results of recent studies show that systemically administered EPO is protective in vivo, in several animal models of neuronal injury. In vitro, EPO prevents neuronal apoptosis induced by a variety of stimuli. This review summarizes the neuroprotective actions of EPO and discusses the underlying mechanisms in terms of signal transduction pathways involved. The understanding of these mechanisms will help differentiate the neuroprotective actions of EPO from its role in the bone marrow.     

Glutamine decreases interleukin-8 and interleukin-6 but not nitric oxide and prostaglandins e(2) production by human gut in-vitro

BACKGROUND: Glutamine modulates cytokine production in various tissues but its effects on the production of other inflammatory mediators such as eicosanoids and nitric oxide have not been investigated in human gut. AIM: To evaluate the influence of glutamine on interleukin (IL)-8, IL-6, nitric oxide and prostaglandin E(2) production by human gut. METHODS: Ten fasted volunteers received either enteral glutamine or isonitrogenous amino acids over 6 h in a cross-over design. Series of duodenal biopsies were frozen or cultured for 24 h with 0.5 or 5 mM of glutamine or amino acids. IL-6, IL-8 and PGE(2) were measured in culture media by ELISA and nitrites by Griess assay. mRNA levels for IL-6, IL-8, Cyclooxygenase-2 and NO synthase-2 were assessed in biopsies by RT-PCR. Results in percent, (median [range]) were compared by Wilcoxon test. RESULTS: Glutamine decreased IL-8 and IL-6 in-vitro production: 63 [2-173] vs 100 [19-177] and 37 [5-489] vs 100 [33-431], both P<0.05. IL-8 mRNA level also decreased in biopsies cultured with 5 mM glutamine: 26 [13-142] vs 92 [34-215], P<0.05. Nitrites and PGE(2) concentrations were not significantly affected by glutamine. CONCLUSION: Glutamine has a specific inhibitory effect on pro-inflammatory cytokine production in the gut and may contribution to the modulation of intestinal inflammation.     

Silibinin attenuates oxidative metabolism and cytokine production by monocytes from preeclamptic women

Abstract

Silibinin is a polyphenolic plant flavonoid with anti-inflammatory properties. The present study investigated the effect of silibinin on oxidative metabolism and cytokine production - tumor necrosis factor-alpha (TNF-α), interleukin (IL)12, granulocyte-macrophage colony-stimulating factor (GM-CSF), IL-6, IL-10, and transforming growth factor beta (TGF-β₁) - by peripheral blood monocytes (PBM) from preeclamptic pregnant women. It is a case-controlled study involving women with preeclampsia (PE, n = 30) compared with normotensive pregnant (NT, n = 30) and with non-pregnant (NP, n = 30) women. Monocytes were obtained and cultured with or without silibinin (5 μM or 50 μM) for 18 h. Superoxide anion (O₂?) and hydrogen peroxide (H₂O₂) release were determined by specific assays, and cytokine levels were determined by immunoenzymatic assays (ELISA). Monocytes from preeclamptic women cultured without stimulus released higher levels of O₂₂, H₂O₂ and TNF-α, and lower levels of IL-10 and TGF-β₁ than did monocytes from NT and NP women. Treatment in vitro with silibinin significantly inhibited spontaneous O₂? and H₂O₂ release and TNF-α production by monocytes from preeclamptic women. The main effect of silibinin was obtained at 50 μM concentration. Thus, silibinin exerts anti-oxidative and anti-inflammatory effects on monocytes from preeclamptic pregnant women by inhibiting the in vitro endogenous release of reactive oxygen species and TNF-α production.     

Expression of PKCeta in NIH-3T3 cells promotes production of the pro-inflammatory cytokine interleukin-6

Protein kinase C encodes a family of enzymes implicated in cellular differentiation, growth control and tumor promotion. The generation and characterization of NIH-3T3 cells which stably overexpress the PKCeta isoform has been previously described by us. In these cells, overexpression of PKCeta altered the expression of specific cell cycle regulators and promoted differentiation [20]. Since PKC has been implicated in the regulation of gene expression, including that of various cytokines, we examined the production of several cytokines in these cells. We report here that out of the major pro-inflammatory cytokines examined, IL-1alpha, IL-1beta, TNF-alpha and IL-6, only IL-6 was generated and secreted in PKCeta -expressing cells without any additional inducer in serum-supplemented cultures (10% FCS). IL-6 was not detected in the control cell line, transfected with the same vector, but lacking the cDNA coding for PKCeta. Moreover, the production of IL-6 on serum stimulation correlated with the levels of PKCeta expressed in these cells. This implies that factors in the serum activate PKCeta and induce IL-6 production. We have examined several growth factors and cytokines for their ability to induce IL-6 production in our PKCeta-expressing cells. Among the growth factors tested (EGF, PDGF, FGF, insulin, IGF-1 and IL-1), PDGF and FGF were the most potent IL-6 inducers. The effects of FGF and PDGF on IL-6 production were blocked in the presence of PKC inhibitors. We also examined the signaling pathways that mediate production of IL-6 in PKCeta-expressing cells. Using specific inhibitors of the MAPK pathway, we have shown a role for ERK and p38 MAPK in FGF- and serum-stimulated IL-6 production, but only for p38 MAPK in PDGF-stimulated IL-6 production. Our studies provide evidence that PDGF and FGF can serve as upstream regulators of PKCeta and that PKCeta is involved in the expression of IL-6. This suggests that inhibition of PKC may provide a basis for the development of drugs for the treatment of disorders in which IL-6 is pathologically involved.     

n-3 PUFA supplementation, monocyte PCA expression and interleukin-6 production

n-3 polyunsaturated fatty acids (PUFA) can affect several monocyte functions and the biochemistry of blood cells, thus possibly influencing the initiation of thrombosis, inflammatory disease and atherosclerosis. In this study, we have investigated the effect of dietary supplementation with n-3 PUFA ethyl esters on procoagulant activity (PCA) and interleukin-6 (IL-6) production by human mononuclear cells. Nine healthy volunteers received 4 g/d of n-3 PUFA ethyl esters (4 × 1 g capsules with at least 85% eicosapentaenoic + docosahexaenoic acid ethyl esters) for 18 weeks. Before and at the end of the treatment, mononuclear cells were obtained from peripheral citrated blood by Ficoll-Hypaque density gradient centrifugation. Cellular suspensions (10⁷ cells/ml) were incubated at 37°C for 4 h in the absence and presence of lipopolysaccharide (10 μg/ml); PCA was determined by one-stage clotting assay and IL-6 concentrations were assayed in supernatants by specific ELISA. After 18-week treatment, both unstimulated and stimulated monocyte PCA were significantly reduced by 66% and 63%, respectively (P < 0.01). Similarly, a significant inhibitory effect by n-3 PUFA treatment on basal and LPS-stimulated IL-6 monocyte production was observed (50% and 46%, respectively, P < 0.05). These data indicate that 18-week n-3 PUFA supplementation may influence monocyte activities, which play a specific role in atherosclerosis and its thrombotic complications.     

Preliminary results on interleukin-4 and interleukin-10 cytokine production in malnourished, inducible nitric oxide synthase-deficient mice with schistosomiasis mansoni infection

Schistosoma mansoni infected C57Bl/6 inducible nitric oxide synthase (iNOS)-deficient and non-deficient malnourished mice, both fed a balanced controlled diet were studied. Interleukins, IL-4 and IL-10 responses to soluble egg antigens (SEA) 90 days after infection, were determined. Our results suggest that in iNOS deficient, malnourished mice, 90 days after of infection, nitric oxide has a downregulating effect on IL-4 and IL-10 production. We are currently investigating the biological significance of these findings.     

Daytime napping after a night of sleep loss decreases sleepiness, improves performance, and causes beneficial changes in cortisol and interleukin-6 secretion

Sleep loss has been associated with increased sleepiness, decreased performance, elevations in inflammatory cytokines, and insulin resistance. Daytime napping has been promoted as a countermeasure to sleep loss. To assess the effects of a 2-h midafternoon nap following a night of sleep loss on postnap sleepiness, performance, cortisol, and IL-6, 41 young healthy individuals (20 men, 21 women) participated in a 7-day sleep deprivation experiment (4 consecutive nights followed by a night of sleep loss and 2 recovery nights). One-half of the subjects were randomly assigned to take a midafternoon nap (1400-1600) the day following the night of total sleep loss. Serial 24-h blood sampling, multiple sleep latency test (MSLT), subjective levels of sleepiness, and psychomotor vigilance task (PVT) were completed on the fourth (predeprivation) and sixth days (postdeprivation). During the nap, subjects had a significant drop in cortisol and IL-6 levels (P < 0.05). After the nap they experienced significantly less sleepiness (MSLT and subjective, P < 0.05) and a smaller improvement on the PVT (P < 0.1). At that time, they had a significant transient increase in their cortisol levels (P < 0.05). In contrast, the levels of IL-6 tended to remain decreased for approximately 8 h (P = 0.1). We conclude that a 2-h midafternoon nap improves alertness, and to a lesser degree performance, and reverses the effects of one night of sleep loss on cortisol and IL-6. The redistribution of cortisol secretion and the prolonged suppression of IL-6 secretion are beneficial, as they improve alertness and performance.     

Supernatants derived from CD8+ lymphocytes activated by mycobacterial fractions inhibit cytokine production. The role of interleukin-6

Our study examined the effects of supernatants derived from CD8+ lymphocytes treated with high molecular weight components ofMycobacterium tuberculosis on cytokine production. Such suppressor but not control supernatants increased the production of IL-4 and IL-6 whilst suppressing IL-1, TNF-alpha, IL-2 and IFN- productionby monocytes andlymphocytes. The effects on cytokine production were time dependent being observed as early as 4 hours with peak activity observed at 24 hours.The inhibition of IL-1 and TNF-alpha by monocytes appeared to be related to increases in IL-6 levels present in supernatants of non-adherent lymphocytes incubated with mycobacterial components. This was confirmed by studies demonstrating that the addition of recombinant IL-6 to cultures depressed the production of these cytokines. Furthermore the addition of monoclonal anti-IL6 to such cultures restored the production of IL-1 and TNF-alpha. The results suggest that mycobacterial components inhibit host cellular functions by manipulating the host's cytokine network.     

Stimulation of interleukin-6 production by corticotropin-releasing factor.

Based on the immune-modulating properties of corticotropin-releasing factor (CRF), the effect of this peptide for interleukin-6 (IL-6) production was investigated. Using human peripheral blood mononuclear cells (MNC), the amount of bioactive IL-6 produced was significantly (P less than or equal to 0.05) increased by CRF (10(-10) to 10(-7) M range). However, the IL-6 production of lipopolysaccharide-treated MNC cultures was not modified. At concentrations of greater than or equal to 10 nM, CRF and two analogous peptides (Tyr-CRF and alpha-helical CRF) elicited 16- to 21-fold stimulation of IL-6 production by MNC. Purified monocytes, but not purified lymphocytes, were the cells that responded to CRF action exhibiting nearly 19-fold stimulation at 100 nM concentration. The CRF-induced production of IL-6 cytokine by peripheral blood MNC may suggest a messenger role for this neurohormone in the feedback control of neuroendocrine-immune circuitry.     

The regulation of fatty acid and branched-chain amino acid oxidation in cancer cachectic rats: a proposed role for a cytokine, eicosanoid, and hormone trilogy

Some postulates of a hypothesis concerned with the deregulation of muscle turnover by the hypoketonemia of cachectic tumor-bearing rats were examined. Plasma concentrations of ketone bodies (D-(3)-hydroxybutyrate + acetoacetate) in rats bearing the Walker 256 carcinosarcoma were reduced by 45% (P less than 0.001) whereas the concentrations of triglyceride and free fatty acids were elevated by 223% (P less than 0.001) and 335% (P less than 0.001), respectively. Parallel with the changes in plasma, the livers of tumor-bearing animals showed decreased concentrations of KB by 35% (P less than 0.05) and increased concentrations of TG and FFA by 49% (NS) and 15% (NS), respectively. In comparison with values for the control liver (fed ad libitum), the perfused liver of animals bearing the Walker 256 tumor formed 42% (P less than 0.05) and 75% (P less than 0.05) less ketone bodies and CO2, respectively, from oleate, while TG formation was enhanced by 33% (P less than 0.001). There was two- to threefold (P less than 0.001) enhancement of [1-14C]leucine oxidation in vivo by the tumor-bearing animals. The activities of branched-chain amino acid aminotransferase and branched-chain keto acid dehydrogenase were elevated by 70% (P less than 0.001) and 560% (P less than 0.001) respectively in the gastrocnemius muscle of the tumor-bearing animals. The results of the investigation supported a second proposal of the hypothesis, namely, that cancer-induced cachexia resulted in the notable elevation in the concentration of arginine vasopressin that was accompanied by parallel increases in the plasma, urine, and muscle concentrations of prostaglandin E2. The proposals of the original hypothesis have been augmented to include roles for PGE2 and the cytokine cachectin/tumor necrosis factor which may engineer all of the events depicted in the original hypothesis.