TNFalpha production in whole blood cultures from healthy individuals

Tumor necrosis factor alpha (TNFalpha) is a major mediator of inflammatory responses and also plays a prominent role in bridging the innate and adaptive phases of immunity. In the present work we attempted to study TNFalpha production in endotoxin-stimulated blood of healthy individuals, and the inter-individual variability in TNFalpha production. For this study, we used diluted whole blood stimulated with lipopolysaccharide (LPS). The levels of the pro-inflammatory cytokine TNFalpha were measured by ELISA and by the L929 cytotoxicity bioassay in 16 and 18 healthy donors, respectively. There were highly significant inter-individual variations in the induced TNFalpha production. It is worth noting that there was no difference in sensitivity between ELISA and the cytotoxicity L929 bioassay. We concluded that whole blood culture is a sensitive method to determine the pro-inflammatory cytokine production in response to endotoxin stimuli in a relevant physiologic milieu. Our data indicate that this method provides appropriate information about the state of cellular immunity of the individual.     

Cytokine production of stimulated whole blood cultures in rheumatoid arthritis patients receiving short-term infliximab therapy

Patients with rheumatoid arthritis (RA) treated with anti-tumor necrosis factor (TNF) strategies have an increased susceptibility to infections, especially those caused by intracellular pathogens. In this study we assessed the cytokine production capacity in patients with RA and we further investigated whether anti-TNF therapy modulates the production of pro-inflammatory cytokines involved in the resistance against infections. Whole blood cultures from 10 RA patients and 10 healthy controls were stimulated with heat-killed Candida albicans, Salmonella typhimurium, Staphyloccocus aureus, Aspergillus fumigatus or Mycobacterium tuberculosis and production of interleukin (IL)-1beta, IL-6, IL-10, interferon (IFN)-gamma and TNF-alpha was measured. Before anti-TNF therapy, whole blood cultures from RA patients released significantly less IFN-gamma than healthy controls after stimulation with all tested microorganisms. Short-term anti-TNF therapy did not have an inhibitory effect on the release of the cytokines tested. We conclude that cells of patients with RA have a strongly reduced production capacity of IFN-gamma after bacterial challenge. Although short-term therapy with anti-TNF agents did not further decrease the release of other proinflammatory cytokines, the combination of defective IFN-gamma production in basal conditions and TNF neutralization during anti-TNF therapy is likely to be responsible for the higher susceptibility to infections in patients with RA.     

A short course of oral aspirin increases IL-18-induced interferon-gamma production in whole blood cultures

The effect of aspirin on whole blood cytokine production was studied in six healthy volunteers. Four days after cessation of a 3-day regimen of 650 mg of oral aspirin, there was a 70% increase in interferon-gamma (IFN-gamma) production, stimulated by a combination of interleukin-18 (IL-18) plus lipopolysaccharide (p < 0.05). At this time, there was a 4-fold increase in the production of tumor necrosis factor-alpha (TNF-alpha) compared to pre-aspirin levels (p < 0.03). TNF-alpha and IFN-gamma production returned to pre-aspirin levels one month after the discontinuation of aspirin. Short-term aspirin treatment induces a significant increase in the production of these cytokines, probably through inhibition of prostaglandins. These data suggest a novel pathway through which long aspirin use reduces the risk of colon cancer, and may explain the effects of aspirin in inflammatory bowel disease.     

Non-infected preterm parturition is related to increased concentrations of IL-6, IL-8 and MCP-1 in human cervix

Background  

Human cervical ripening is an inflammatory process. In labour at term the mRNA-levels and protein concentrations for interleukin-6 (IL-6) and IL-8 in cervix significantly increase. The aim of this study was to investigate if there are differences in the inflammatory process of preterm and term cervical ripening.     

Modification of cord blood IL-6 production with IgM enriched human immunoglobulin in term and preterm infants

Pro-inflammatory cytokines contribute significantly to the morbidity of premature infants. IL-6 and IL-8 are involved in the pathogenesis of pulmonary and cerebral tissue injury. The effect of human immunoglobulin preparations on cytokine production in preterm infants has not been studied. We investigated the influence of immunoglobulin on LPS stimulated IL-6 and IL-8 production in cord blood of healthy preterm neonates. Ten non-infected preterm infants delivered by cesarean section and 5 healthy term neonates were included. In the preterm infants, significant IL-6 production was observed in the absence of immunoglobulin after 4 h [median 113 (39-725) pg/ml], 8 h [375 (234-1795) pg/ml] and 12 h [360 (248-2765) pg/ml] of LPS incubation. IL-6 concentrations were significantly lower after incubation with LPS+immunoglobulin after 4 h [median 38 (5-568) pg/ml; p=0.005], 8 h [178 (10-1830) pg/ml; p=0.001] and 12 h [182 (29-2530) pg/ml; p=0.002]. Cultures from term infants produced IL-6 levels approx. 4 times of those from premature infants unaffected by immunoglobulin. IL-8 production also correlated to gestational age and was not affected by immunoglobulin in both groups. Human immunoglobulin preparation may modify IL-6 production in cord blood cultures from premature infants.     

IL-29 enhances Toll-like receptor-mediated IL-6 and IL-8 production by the synovial fibroblasts from rheumatoid arthritis patients

Introduction

We previously reported that IL-29, a newly described member of interferon (IFN) family, was overexpressed in blood and synovium of rheumatoid arthritis (RA) patients and triggered proinflammatory cytokine IL-6 and IL-8 mRNA expression in RA synovial fibroblasts (RA-FLS). This suggests that IL-29 has an important role in synovial inflammation. Toll-like receptors (TLRs) also activate RA-FLS to produce inflammatory mediators including tumor necrosis factor α (TNF-α) and IL-1β in RA-FLS. Since the TLR family plays an early role in the innate immune response and the subsequent induction of the adaptive immune response, we hypothesize that IL-29 interacts with TLRs in RA inflammation. This study aimed to investigate the effect of IL-29 on TLR-mediated proinflammatory cytokine production in RA-FLS.

Methods

The mRNA level of IL-29 receptors (IL-28Rα and IL-10R2) in RA-FLS was determined by semi-quantitative RT- PCR. IL-6 and IL-8 mRNA expressions in RA-FLS were evaluated by real-time PCR after pre-incubation with IL-29 and subsequent stimulation with peptidoglycan (PGN, TLR2 ligand), or polycytidylic acid (poly(I:C), TLR3 ligand), or lipopolysaccharide (LPS, TLR4 ligand) . The production of TLR2, 3, and 4 in RA-FLS after IL-29 stimulation was also assessed by real-time PCR and flow cytometry. IL-29 mRNA and protein expression in RA-FLS after stimulation with PGN, poly(I:C), or LPS were measured by real-time PCR and enzyme-linked immunosorbent assay (ELISA), respectively.

Results

The IL-29 receptor complex (IL-28Rα and IL-10R2) was identified in RA-FLS. IL-29 enhanced TLR-mediated IL-6 and IL-8 expression in RA-FLS. IL-29 upregulated expression of TLR2, 3 and 4 in RA-FLS. Exposure to PGN, poly(I:C) or LPS triggered IL-29 production by RA-FLS.

Conclusions

We show for the first time that IL-29 enhances TLR-induced proinflammatory cytokine production in RA-FLS via upregulation of TLRs.     

Methemoglobin is a potent activator of endothelial cells by stimulating IL-6 and IL-8 production and E-selectin membrane expression

Infection and injury are frequently accompanied by hemolysis. Endothelial cells are direct targets of free Hb or its oxidative derivatives, including methemoglobin (MHb) and hemin. This study tested whether Hb or its derivatives alter chemokine (IL-8) and cytokine (IL-6) production and the membrane expression of cell adhesion molecule (E-selectin) in human umbilical vein endothelial cells (passages 2-4, HUVECs). E-selectin membrane content and IL-6 and IL-8 release were quantified by ELISA; cellular mRNA levels were determined by RT-PCR. MHb in vitro resulted in a dose (1-50 µM)- and time (2-16 h)-dependent increase in E-selectin membrane content and IL-6 and IL-8 release in HUVECs. The stimulatory effect of MHb (12 µM) on E-selectin membrane expression and IL-6 and IL-8 release was similar to that produced after treatment with TNF- (5 ng/ml) and IL-1 (0.25 ng/ml). In contrast, Hb or hemin had no effects. As expected, MHb, Hb, and hemin markedly induced heme oxygenase-1 expression in HUVECs. Haptoglobin, cytochalasin D, and actinomycin inhibited the MHb-induced responses, whereas zinc protoporphyrin IX (a heme oxygenase inhibitor) or desferroxamine (an iron chelator) did not inhibit MHb-induced responses. MHb also increased cellular mRNA levels of E-selectin, IL-6, and IL-8. MHb treatment activated cellular NF-B and NF-B inhibitors; N-acetyl cysteine, SN50, and caffeic acid phenylethyl ester inhibited the MHb-induced responses. These data indicate that MHb is a potent activator of endothelial cells through NF-B-mediated upregulation of cell adhesion molecule expression and chemokine and cytokine production. MHb-induced endothelial cell activation may have clinical significance after infections, hemolysis, or methemoglobinemia. human umbilical vein endothelial cells; cytokine; chemokine; adhesion molecule; hemolysis; hemoglobin; hemin; nuclear factor-B     

Chemokines regulate IL-6 and IL-8 production by fibroblast-like synoviocytes from patients with rheumatoid arthritis.

Rheumatoid arthritis (RA) is characterized by proliferation of synoviocytes that produce inflammatory cytokines and chemokines. The expressed chemokines are thought to be involved in the migration of inflammatory cells into the synovium. In this study we show that CCL2/monocyte chemotactic protein-1, CCL5/RANTES, and CXCL12/stromal cell-derived factor-1 enhanced IL-6 and IL-8 production by fibroblast-like synoviocytes (FLS) from patients with RA, and their corresponding receptors, CCR2, CCR5, and CXCR4, respectively, were expressed by RA FLS. The chemokines stimulated RA FLS more effectively than skin fibroblasts. Culture with CCL2 enhanced phosphorylation of extracellular signal-related kinase 1 (ERK1) and ERK2, but not phosphorylation of p38 or Src. Moreover, activation of ERK1/2 was inhibited by pertussis toxin, a G(i)-coupled protein inhibitor, and RS-504393, CCR2 antagonist, suggesting that ERK1/2 was activated by CCL2 via CCR2 and G(i)-coupled protein. On the other hand, CCL2, CCL5, and CXCL12 were expressed on RA FLS, and their production was regulated by TNF-alpha, IL-1beta, and TGF-beta1. Our results indicate that the chemokines not only play a role in inflammatory cell migration, but are also involved in the activation of FLS in RA synovium, possibly in an autocrine or paracrine manner.     

Impaired production of interleukin 6 and tumour necrosis factor alpha in whole blood cell cultures of patients with multiple myeloma

Cytokine production in whole blood cell cultures can be an indicator of immune cellular status in neoplastic patients. Production of interleukin 6 (IL-6) and tumour necrosis factor alpha (TNF-alpha) after stimulation with lipopolysaccharide was measured in 1/10 diluted whole blood culture of patients with monoclonal gammopathies [monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma (MM)]. With this system, lower production of IL-6 and TNF-alpha at 4, 24 and 48 h in total blood cultures in patients with symptomatic MM was observed. Thus, the capacity of cytokine production was greater in control subjects and in patients with MGUS and MM in response than in symptomatic MM (P=0.005 at 4 h and P=0. 006 at 24 and 48 h for IL-6 and P<0.0001 at 24 h and P<0.001 at 48 h for TNF-alpha). These differences remained significant after adjustment on the basis of the lymphocyte count (P<0.001 for IL-6 and TNF-alpha at 24 and 48 h). The impaired IL-6 and TNF-alpha production in patients with symptomatic MM is probably due to a tumour-related immunosuppressive status.     

Ionizing radiation enhances IL-6 and IL-8 production by human endothelial cells

Irradiation exposure is known to induce an inflammatory reaction. Endothelial cells play a crucial role both in the inflammatory process and in radiation damage. Therefore, supernatants and cell lysates of (60)Co-irradiated human umbilical vein endothelial cells (HUVEC) have been assessed for the presence of pro-inflammatory cytokines. After gamma irradiation, interleukin (IL)-1alpha, IL-1beta and tumor necrosis factor (TNF)-alpha remained undetectable in both cell supernatants and cell lysates. However, a dose-dependent increase in the production of IL-6 and IL-8 has been demonstrated up to 6 days after exposure. These data indicate that the pro-inflammatory cytokines IL-6 and IL-8 may be involved in the inflammatory response of vascular endothelium induced by exposure to ionizing radiation.     

Thixotropic properties of whole blood from healthy human subjects

The steady state non-Newtonian viscosity of whole human blood has been widely studied as a function of the shear rate; and used to characterize the blood in various pathological disorders. In our previous studies, we demonstrated that blood is a thixotropic fluid. Its time-dependency and shear rate dependency of rheological behavior can be represented by an equation developed by Huang. Parameters of the equation can be used for the characterization of an individual's blood. They provide information, such as the kinetic rate constant of breakdown of RBC rouleaux to individual erythrocytes and the relative amount of rouleau formation in the dynamic equilibrium between rouleaux and individual erythrocytes. In this communication, the thixotropic parameters from blood samples of fifteen apparently healthy human subjects were investigated. When compared to the use of apparent viscosity values for the correlation with a pathological disorder, thixotropic parameters are preferable. The mean values of thixotropic parameters obtained from apparently healthy human subjects provide a base for comparison with the same parameters as obtained from blood samples of patients with certain pathological disorders involving the circulation.     

Comparison of cytokine production in cultures of whole human blood and purified mononuclear cells.

In order to examine inter- and intra-individual variations in cytokine production, blood was collected from 48 healthy subjects on each of 4 occasions separated by 4 weeks. Whole blood (diluted 1:10) and mononuclear cell (MNC) cultures were stimulated for 24 h with either concanavalin A (Con A) or bacterial lipopolysaccharide (LPS) and the concentrations of IL-1alpha, IL-1beta, IL-2, TNF-alpha, IL-10 and IFN-gamma in the culture medium measured. There were highly significant inter-individual variations in the production of each of the cytokines measured. However, the level of the production of each cytokine appeared to be characteristic of an individual. There were significant correlations between production of each cytokine in whole blood and MNC cultures. It is concluded that there is significant inter-individual variation in cytokine production which is unaffected by time or by the stimulus used to elicit cytokine production, and that whole blood cultures can be used instead of MNC cultures to measure cytokine production.     

IL-6 stimulates osteoclast-like multinucleated cell formation in long term human marrow cultures by inducing IL-1 release

IL-6 enhances the differentiation of pluripotent hematopoietic stem cells but predominantly affects the differentiation of hematopoietic cells in the granulocyte-macrophage lineage. We have previously shown that multinucleated cells (MNC) with many features of the osteoclast phenotype form in long term human marrow cultures. Addition of rhIL-6 (10 to 100 pg/ml) to these cultures significantly increased MNC formation, with greater than 80% of the MNC expressing an Ag that cross-reacts with the mAb 23c6. This antibody preferentially binds to osteoclasts. rhIL-6 did not enhance MNC formation in marrow cultures treated with 1,25 dihydroxyvitamin D3, a potent stimulator of MNC formation, but significantly increased the percentage of MNC that cross-reacted with the 23c6 mAb. Addition of antihuman IL-1 to cultures treated with rhIL-6 totally inhibited the increase in MNC formation stimulated by rhIL-6. In contrast, anti-IL-1 did not affect MNC formation stimulated by 1,25 dihydroxyvitamin D3. Further, conditioned media from marrow cultures exposed to rhIL-6 contained elevated levels of IL-1 beta (500 pg/ml compared to 23 pg/ml in control cultures 15 h after IL-6 addition). These results suggest that the capacity of rhIL-6 to stimulate formation of MNC which cross-react with 23c6 is mediated by induction of release of IL-1 beta.     

Intestinal permeability is reduced and IL-10 levels are increased in septic IL-6 knockout mice

Sepsis is associated with increased intestinal permeability, but mediators and mechanisms are not fully understood. We examined the role of interleukin (IL)-6 and IL-10 in sepsis-induced increase in intestinal permeability. Intestinal permeability was measured in IL-6 knockout (IL-6 -/-) and wild-type (IL-6 +/+) mice 16 h after induction of sepsis by cecal ligation and puncture or sham operation. In other experiments, mice or intestinal segments incubated in Ussing chambers were treated with IL-6 or IL-10. Intestinal permeability was assessed by determining the transmucosal transport of the 4.4-kDa marker fluorescein isothiocyanate conjugated dextran and the 40-kDa horseradish peroxidase. Intestinal permeability for both markers was increased in septic IL-6 +/+ mice but not in septic IL-6 -/- mice. Treatment of nonseptic mice or of intestinal segments in Ussing chambers with IL-6 did not influence intestinal permeability. Plasma IL-10 levels were increased in septic IL-6 -/- mice, and treatment of septic mice with IL-10 resulted in reduced intestinal permeability. Increased intestinal permeability during sepsis may be regulated by an interaction between IL-6 and IL-10. Treatment with IL-10 may prevent the increase in mucosal permeability during sepsis.     

The relationship between ultrasonographically measured testicular volumes and cord blood inhibin B concentrations in healthy term male neonates

Serum inhibin B (INHB) concentrations are associated with testicular volumes (TV) in all periods of childhood. The aim of the study was to investigate the relationship between TV measured by ultrasonography (US) and cord blood inhibin B and total testosterone (TT) concentrations, stratified by mode of delivery. In total 90 male infants were included. Testes of healthy, term newborns were evaluated by US on the third day after delivery. TV were calculated using two formulae: The ellipsoid formula [length (mm) × width (mm²) × π/6] and Lambert formula [length (mm) x width (mm) x height (mm) x 0.71]. Cord blood was taken for the determination of total testosterone (TT) and INHB. TT and INHB concentrations were evaluated according to TV percentiles (<10th, 10th-90th, >90th). There was a strong positive correlation between mean TV calculated with both formulae by percentile group (r = 0.777, r = 0.804, r = 0.846; p < 0.001). Cord blood INHB, but not TT were significantly lower in newborns with TV < 10th percentile compared to those with TV between 10 and 90th percentile and > 90th percentile (p < 0.05). There was a positive correlation between left and right TV calculated by either formula, and cord blood INHB (r = 0.212, 0.313, 0.320, 0.246, p < 0.05), not TT. There was no significant difference between hormones and TV when grouped by mode of delivery (p > 0.05). The Lambert and ellipsoid formulas are equally reliable in calculating neonatal testicular by ultrasound. INHB concentration is high in cord blood and positively correlated with neonatal TV. Cord blood INHB concentration may be an indicator for early detection of testicular structure and function disorders in neonates.     

The role of p38 mitogen-activated protein kinase in IL-6 and IL-8 production from the TNF-alpha- or IL-1beta-stimulated rheumatoid synovial fibroblasts

We examined the role of p38 mitogen-activated protein (MAP) kinase in the tumor necrosis factor alpha (TNF-alpha)- or interleukin-1beta (IL-1beta)-induced production of interleukin-6 (IL-6) and interleukin-8 (IL-8) in fresh rheumatoid synovial fibroblast (RSF) cultures concomitantly with the induction of p38 MAP kinase activity. Pretreatment of RSF with a specific p38 MAP kinase inhibitor, SB203580, blocked the induction of IL-6 and IL-8 without affecting nuclear translocation of nuclear factor kappaB (NF-kappaB) or IL-6 and IL-8 mRNA levels. These findings suggest that p38 MAP kinase inhibitor may have synergistic, rather than additive, effect for the treatment of rheumatoid arthritis.