In vitro release of interleukin-15 by broncho-alveolar lavage cells and peripheral blood mononuclear cells from patients with different lung diseases

IL-15 shares several biological activities with IL-2 and uses the b and g chain of the IL-2 receptor. In addition to its T-cell stimulating capacity, IL-15 exhibits regulatory properties on macrophage proinflammatory cytokine release. IL-15 is released by non-lymphoid cells, e.g. muscle cells, fibroblasts and monocytes/macrophages. In many lung diseases alveolar macrophages (AM) are activated and release pro- inflammatory cytokines. We asked whether IL-15 is released ex vivo by AM and peripheral blood mononuclear cells (PBMC) from patients with inactive sarcoidosis (PSi), active sarcoidosis (PSa), tuberculosis (TB), hypersensitivity pneumonitis (HSP), cryptogenic fibrosing alveolitis (CFA) and pneumonia (PN). Additionally, we examined the kinetics of the IL-15 release of these cells. During 24 hours of culture, AM from controls (CO) released 3.8 +/- 1.9 pg/ml (mean +/- SD) of IL-15, which was significantly lower than in most of the patient groups (PSa: 8.7 +/- 3.9 pg/ml, TB: 8.4 +/- 1.9 pg/ml, CFA: 5.7 +/- 1.5 pg/ml, and PN: 7. 8 +/- 2.6 pg/ml) except PSi (4.0 +/- 2.6 pg/ml) and HSP (9.3 +/- 9.5 pg/ml). PBMC from patients with PSa released significantly more IL-15 than PBMC from CO (10.8 +/- 8.9 pg/ml versus 6.9 +/- 2.2 pg/ml) whereas PBMC IL-15 release of the other groups did not differ from CO (TB: 5.7 +/- 1.4 pg/ml; CFA: 4.6 +/- 1.6 pg/ml; HSP: 4.9 +/- 3.8 pg/ml). Kinetic studies revealed a minor peak after 5 hours and a major peak from 12 hours to 35 hours for AM and PBMC. In summary, AM from all patient groups but the PSi and the HSP group released increased levels of IL-15, although the total amount of this cytokine is very low.     

Interleukin-6 impairs endothelium-dependent NO-cGMP-mediated relaxation and enhances contraction in systemic vessels of pregnant rats

IL-6 is elevated in plasma of preeclamptic women, and twofold elevation of plasma IL-6 increases vascular resistance and arterial pressure in pregnant rats, suggesting a role of the cytokine in hypertension of pregnancy. However, whether the hemodynamic effects of IL-6 reflect direct effects of the cytokine on the mechanisms of vascular contraction/relaxation is unclear. The purpose of this study was to test the hypothesis that IL-6 directly impairs endothelium-dependent relaxation and enhances vascular contraction in systemic vessels of pregnant rats. Active stress was measured in aortic strips isolated from virgin and late pregnant Sprague-Dawley rats and then nontreated or treated for 1 h with IL-6 (10 pg/ml to 10 ng/ml). In endothelium-intact vascular strips, phenylephrine (Phe, 10(-5) M) caused an increase in active stress that was smaller in pregnant (4.2 +/- 0.3) than virgin rats (5.1 +/- 0.3 x 10(4) N/m(2)). IL-6 (1,000 pg/ml) caused enhancement of Phe contraction that was greater in pregnant (10.6 +/- 0.7) than virgin rats (7.5 +/- 0.4 x 10(4) N/m(2)). ACh and bradykinin caused relaxation of Phe contraction and increases in vascular nitrite production that were greater in pregnant than virgin rats. IL-6 caused reductions in ACh- and bradykinin-induced vascular relaxation and nitrite production that were more prominent in pregnant than virgin rats. Incubation of endothelium-intact strips in the presence of N(omega)-nitro-L-arginine methyl ester (10(-4) M) to inhibit nitric oxide (NO) synthase, or 1H-[1,2,4]oxadiazolo[4,3]-quinoxalin-1-one (ODQ, 10(-5) M) to inhibit cGMP production in smooth muscle, inhibited ACh-induced relaxation and enhanced Phe-induced stress in nontreated but to a lesser extent in IL-6-treated vessels, particularly those of pregnant rats. Removal of the endothelium enhanced Phe-induced stress in nontreated but not IL-6-treated vessels, particularly those of pregnant rats. In endothelium-denuded strips, relaxation of Phe contraction with sodium nitroprusside, an exogenous NO donor, was not different between nontreated and IL-6-treated vessels of virgin or pregnant rats. Thus IL-6 inhibits endothelium-dependent NO-cGMP-mediated relaxation and enhances contraction in systemic vessels of virgin and pregnant rats. The greater IL-6-induced inhibition of vascular relaxation and enhancement of contraction in systemic vessels of pregnant rats supports a direct role for IL-6 as one possible mediator of the increased vascular resistance associated with hypertension of pregnancy.     

Interleukin-2 enhances the release of endothelin-1 from the rat mesenteric artery

We measured the ET-1 concentration in plasma and in the perfusate of the mesenteric arteries of rats treated with a therapeutic dose of IL-2 for 7 days (100000 U/Kg, iv.). The plasma ET-1 concentration in rats given IL-2 was 14.2 +/- 3.2 pg/ml which was significantly greater than that in the controls (2.5 +/- 0.4 pg/ml, P less than 0.05). The mesenteric arteries also released a significantly greater amount of ET-1 (29.5 +/- 1.6 pg/h) than that in controls (16.8 +/- 2.3 pg/h, P less than 0.01). The arterial blood pressure was significantly lower after IL-2 treatment than the pre-dosing level (P less than 0.05). It is concluded that IL-2 induces ET-1 release from the vascular wall, possibly as a result of reversible endothelial dysfunction caused by IL-2.     

Non-Invasive Technology That Improves Cardiac Function after Experimental Myocardial Infarction: Whole Body Periodic Acceleration (pGz)

Myocardial infarction (MI) may produce significant inflammatory changes and adverse ventricular remodeling leading to heart failure and premature death. Pharmacologic, stem cell transplantation, and exercise have not halted the inexorable rise in the prevalence and great economic costs of heart failure despite extensive investigations of such treatments. New therapeutic modalities are needed. Whole Body Periodic Acceleration (pGz) is a non-invasive technology that increases pulsatile shear stress to the endothelium thereby producing several beneficial cardiovascular effects as demonstrated in animal models, normal humans and patients with heart disease. pGz upregulates endothelial derived nitric oxide synthase (eNOS) and its phosphorylation (p-eNOS) to improve myocardial function in models of myocardial stunning and preconditioning. Here we test whether pGz applied chronically after focal myocardial infarction in rats improves functional outcomes from MI. Focal MI was produced by left coronary artery ligation. One day after ligation animals were randomized to receive daily treatments of pGz for four weeks (MI-pGz) or serve as controls (MI-CONT), with an additional group as non-infarction controls (Sham). Echocardiograms and invasive pressure volume loop analysis were carried out. Infarct transmurality, myocardial fibrosis, and markers of inflammatory and anti-inflammatory cytokines were determined along with protein analysis of eNOS, p-eNOS and inducible nitric oxide synthase (iNOS).At four weeks, survival was 80% in MI-pGz vs 50% in MI-CONT (p< 0.01). Ejection fraction and fractional shortening and invasive pressure volume relation indices of afterload and contractility were significantly better in MI-pGz. The latter where associated with decreased infarct transmurality and decreased fibrosis along with increased eNOS, p-eNOS. Additionally, MI-pGz had significantly lower levels of iNOS, inflammatory cytokines (IL-6, TNF-α), and higher level of anti-inflammatory cytokine (IL-10). pGz improved survival and contractile performance, associated with improved myocardial remodeling. pGz may serve as a simple, safe, non-invasive therapeutic modality to improve myocardial function after MI.     

In vivo upregulation of nitric oxide synthases in healthy rats

Periodic acceleration (pGz), sinusoidal motion of the whole body in a head–foot direction in the spinal axis, is a novel noninvasive means for cardiopulmonary support and induction of pulsatile shear stress. pGz increases plasma nitrite levels, in vivo and in vitro. Additionally, pGz confers cardioprotection in models of ischemia reperfusion injury. We hypothesize that pGz may also confer a cardiac phenotypic change by upregulation of the expression of the various NO synthase (NOS) isoforms in vivo. pGz was applied for 1 h to awake restrained male rats at 2 frequencies (360 and 600 cpm) and acceleration (Gz) of ±3.4 m/s². pGz did not affect arterial blood gases or electrolytes. pGz significantly increased total nitrosylated protein levels, indicating increased NO production. pGz also increased mRNA and protein levels of eNOS and nNOS, and phosphorylated eNOS in heart. pGz increased Akt phosphorylation (p-AKT), but not total Akt, or phosphorylated ERK1/2. Inducible (i) NOS levels were undetectable with or without pGz. Immunoblotting revealed the localization of nNOS, exclusively in cardiomyocyte, and pGz increased its expression. We have demonstrated that pGz changes myocardial NOS phenotypes. Such upregulation of eNOS and nNOS was still evident 24 h after pGz. Further studies are needed to understand the biochemical and biomechanical signal transduction pathway for the observed NOS phenotype changed induced by pGz.     

Whole Body Periodic Acceleration Is an Effective Therapy to Ameliorate Muscular Dystrophy in mdx Mice

Duchenne muscular dystrophy (DMD) is a genetic disorder caused by the absence of dystrophin in both skeletal and cardiac muscles. This leads to severe muscle degeneration, and dilated cardiomyopathy that produces patient death, which in most cases occurs before the end of the second decade. Several lines of evidence have shown that modulators of nitric oxide (NO) pathway can improve skeletal muscle and cardiac function in the mdx mouse, a mouse model for DMD. Whole body periodic acceleration (pGz) is produced by applying sinusoidal motion to supine humans and in standing conscious rodents in a headward-footward direction using a motion platform. It adds small pulses as a function of movement frequency to the circulation thereby increasing pulsatile shear stress to the vascular endothelium, which in turn increases production of NO. In this study, we examined the potential therapeutic properties of pGz for the treatment of skeletal muscle pathology observed in the mdx mouse. We found that pGz (480 cpm, 8 days, 1 hr per day) decreased intracellular Ca²⁺ and Na⁺ overload, diminished serum levels of creatine kinase (CK) and reduced intracellular accumulation of Evans Blue. Furthermore, pGz increased muscle force generation and expression of both utrophin and the carboxy-terminal PDZ ligand of nNOS (CAPON). Likewise, pGz (120 cpm, 12 h) applied in vitro to skeletal muscle myotubes reduced Ca²⁺ and Na⁺ overload, diminished abnormal sarcolemmal Ca²⁺ entry and increased phosphorylation of endothelial NOS. Overall, this study provides new insights into the potential therapeutic efficacy of pGz as a non-invasive and non-pharmacological approach for the treatment of DMD patients through activation of the NO pathway.     

Plasma proinflammatory cytokine response to surgical stress in elderly patients

We investigated the change of plasma cytokines concentrations in elderly patients during lower abdominal surgery. Plasma interleukin (IL-)6 concentrations (68.0+/-15.4 and 36.1+/-20.7 pg/ml) in elderly patients at 24 h and at 3 days after surgery were significantly higher than those (35.1+/-21.5 and 18.6+/-10.6 pg/ml) of young adults. Plasma IL-6 concentrations (92.3+/- 31.9 pg/ml) in elderly patients anesthetized with propofol and fentanyl were significantly higher at the end of the operation than that (57.9+/-36.7 pg/ml) of elderly patients anesthetized with sevoflurane and fentanyl. In conclusion, elderly patients have an increased and delayed IL-6 response to surgical trauma compared with young adults. Plasma IL-6 production after surgical trauma in elderly patients with total intravenous anesthesia with propofol was significantly higher than that in elderly patients with sevoflurane anesthesia.     

Shear stress-induced vasodilation in porcine coronary conduit arteries is independent of nitric oxide release

The present study was performed to determine the importance of nitric oxide in eliciting epicardial coronary artery dilation during sustained increases in shear stress in the absence of pulsatile flow. Isolated first-order porcine epicardial coronary conduit arteries (approximately 500 microm) were preconstricted (U-46619) and subjected to steady-state changes in flow in vitro. Nonpulsatile flow (shear stress range from 0 to approximately 100 dyn/cm2) produced a graded dilation of epicardial arteries. Inhibiting nitric oxide synthase with 10(-5) M N(omega)-nitro-L-arginine methyl ester (L-NAME) blocked bradykinin-induced vasodilation but did not affect the flow-diameter relation or the maximum change in diameter from static conditions (67 +/- 10 microm in control vs. 71 +/- 8 microm after L-NAME, P = not significant). The addition of indomethacin (10(-5) M) had no effect on flow-mediated vasodilation. Depolarizing vascular smooth muscle with KCl (60 mM) or removing the endothelium blocked bradykinin vasodilation and completely abolished flow-mediated responses. The K+ channel blocker tetraethylammonium chloride (TEA; 10(-4)M) attenuated flow-mediated vasodilation (maximum diameter change was 110 +/- 18 microm under control conditions vs. 58 +/- 10 microm after TEA, P < 0.001). These data indicate that epicardial coronary arteries dilate to steady-state changes in nonpulsatile flow via a mechanism that is independent of nitric oxide production. The ability to completely block this with KCl and attenuate it with TEA supports the hypothesis that epicardial coronary arteries dilate to steady levels of shear stress through hyperpolarization of vascular smooth muscle. This may be secondary to the release of an endothelium-dependent hyperpolarizing factor.     

Periodic acceleration: effects on vasoactive, fibrinolytic, and coagulation factors.

Cellular and isolated vessel experiments have shown that pulsatile and laminar shear stress to the endothelium produces significant release of mediators into the circulation. Periodic acceleration (pG(z)) applied to the whole body in the direction of the spinal axis adds pulses to the circulation, thereby increasing pulsatile and shear stress to the endothelium that should also cause release of mediators into the circulation. The purpose of this study was to determine whether addition of pulses to the circulation through pG(z) would be sufficient to increase shear stress in whole animals and to acutely release mediators and how such a physical maneuver might affect coagulation factors. Randomized control experiments were performed on anesthetized, supine piglets. The treatment group (pG(z)) (n = 12) received pG(z) with a motion platform that moved them repetitively head to foot at +/-0.4 g at 180 cpm for 60 min. The control group (n = 6) was secured to the platform but remained on conventional ventilation throughout the 4-h protocol. Compared with control animals and baseline, pulsatile stress produced significant increases of serum nitrite, prostacyclin, PGE(2), and tissue plasminogen activator antigen and activity, as well as D-dimer. There were no significant changes in epinephrine, norepinephrine, cortisol, and coagulation factors between groups or from baseline values. Pulsatile and laminar shear stress to the endothelium induced by pG(z) safely produces increases of vasoactive and fibrinolytic activity. pG(z) has potential to achieve mediator-related benefits from the actions of nitric oxide and prostaglandins.     

Enhanced plasma IL-6 and IL-1ra responses to repeated vs. single bouts of prolonged cycling in elite athletes.

The impact of repeated bouts of exercise on plasma levels of interleukin (IL)-6 and IL-1 receptor antagonist (IL-1ra) was examined. Nine well-trained men participated in four different 24-h trials: Long [two bouts of exercise, at 0800-0915 and afternoon exercise (Ex-A), separated by 6 h]; Short (two bouts, at 1100-1215 and Ex-A, separated by 3 h); One (single bout performed at the same Ex-A as second bout in prior trials); and Rest (no exercise). All exercise bouts were performed on a cycle ergometer at 75% of maximal O(2) uptake and lasted 75 min. Peak IL-6 observed at the end of Ex-A was significantly higher in Short (8.8 +/- 1.3 pg/ml) than One (5.2 +/- 0.7 pg/ml) but not compared with Long (5.9 +/- 1.2 pg/ml). Peak IL-1ra observed 1 h postexercise was significantly higher in Short (1,774 +/- 373 pg/ml) than One (302 +/- 53 pg/ml) but not compared with Long (1,276 +/- 451 pg/ml). We conclude that, when a second bout of endurance exercise is performed after only 3 h of recovery, IL-6 and IL-1ra responses are elevated. This may be linked to muscle glycogen depletion.     

Periodic acceleration (pGz) prior to whole body Ischemia reperfusion injury provides early cardioprotective preconditioning

AimsPeriodic acceleration (pGz) is a method that applies repetitive sinusoidal head-to-foot motion to the horizontally positioned body. pGz adds pulses to the circulation as a function of frequency, thereby increasing shear stress to the endothelium. Pulsatile shear stress increases release of cardioprotective endothelial-derived nitric oxide prostaglandin E-2 and prostacyclin into the circulation. We investigated whether pGz may be effective as an early preconditioning strategy when applied one hour prior to whole body ischemia reperfusion injury induced by ventricular fibrillation (VF).Main methodsTwenty anesthetized and paralyzed male swine were randomized to one hour of pGz and conventional mechanical ventilation [PC] or solely conventional mechanical ventilation [Control] prior to VF and resuscitation. After eight minutes of unsupported VF, cardiopulmonary resuscitation was carried out followed by defibrillation. Hemodynamics, electrocardiogram, echocardiogram, regional blood flows, and markers of global myocardial injury were measured. Protein expression of endothelial-derived nitric oxide synthase (eNOS), phosphorylated eNOS (p-eNOS), serine/threonine kinase Akt total (t-Akt), and phosphorylated (p-Akt) were determined by immunoblotting.Key findingsAll animals had spontaneous return of circulation after cardiopulmonary resuscitation (CPR) and defibrillation. Preconditioned animals had less hemodynamically significant arrhythmias, less myocardial stunning, and greater regional blood flows to the brain, heart, kidneys, and ileum than Controls. Troponin I and creatine phosphokinase values in PC were 65% of the values present in Controls. In addition, preconditioned animals had higher protein expression of cardiac eNOS, p-eNOS, t-Akt, and p-Akt than Controls.SignificancepGz preconditioning confers early cardioprotection in a model of whole body ischemia reperfusion injury.     

Subcutaneous adipose tissue exerts proinflammatory cytokines after minimal trauma in humans

Inflammatory cytokines released from adipose tissue play an important role in different pathological processes. In the present study, we investigated the inflammatory cytokine response of human subcutaneous adipose tissue (SAT) by applying the open-flow microperfusion technique. Four standard 18-gauge microperfusion catheters were inserted into periumbilical SAT of eight healthy male volunteers [29 +/- 3 yr, BMI 24.3 +/- 1.9 (mean +/- SD)]. SAT probe effluents were collected at 60-min intervals for 8 h after catheter insertion. Different perfusion fluids were used to measure the local effect of insulin and/or glucose on the cytokine response. SAT probe effluents were analyzed for IL-1beta, IL-6, CXCL8 (IL-8), and TNF-alpha. SAT concentrations of IL-1beta increased 100-fold from 1.0 +/- 0.2 pg/ml (mean +/- SE) to 101.5 +/- 23.2 pg/ml (P < 0.001) after 8 h. A 130-fold increase was observed for CXCL8, from 49 +/- 29 to 6,554 +/- 1,713 pg/ml (P < 0.001). Furthermore, a 20-fold increase of IL-6 was observed within the first 5 h (from 159 +/- 123 to 3,554 +/- 394 pg/ml; P < 0.001), and a significant decline to 2,154 +/- 216 pg/ml (P < 0.01) was seen thereafter. Finally, TNF-alpha increased from 1.4 +/- 0.6 to 2.5 +/- 0.5 pg/ml (P < 0.05) in hour 2 and remained stable thereafter. Local administration of insulin exerted a stimulatory effect on the inflammatory response of IL-6. In conclusion, SAT exerts a highly reproducible and consistent proinflammatory cytokine response after minimally invasive trauma caused by the insertion of a catheter in humans.     

Identification of a novel cytokine, ML-1, and its expression in subjects with asthma

A novel gene, designated ML-1, was identified from a human genomic DNA clone and human T cell cDNA sequences. The second exon of ML-1 gene shares significant sequence identity with the gene encoding IL-17 (IL-17). ML-1 gene expression was up-regulated in activated PBMCs, CD4(+) T cells, allergen-specific Th0, Th1, and Th2 clones, activated basophils, and mast cells. Increased expression of the ML-1 gene, but not IL-17, was seen following allergen challenge in four asthmatic subjects, suggesting its role in allergic inflammatory responses. ML-1 from transiently transfected COS-7 cells was able to induce gene expression and protein production for IL-6 and IL-8 (at 10 ng/ml of ML-1: for IL-6, 599.6 +/- 19.1 pg/ml; for IL-8, 1724.2 +/- 132.9 pg/ml; and at 100 ng/ml of ML-1: for IL-6, 1005.3 +/- 55.6 pg/ml; for IL-8, 4371.4 +/- 280.5 pg/ml; p < 0.05 for both doses vs baseline) in primary bronchial epithelial (PBE) cells. Furthermore, increased expression of ICAM-1 was found in ML-1-stimulated PBE cells (mean fluorescence intensity (MFI) = 31.42 +/- 4.39 vs baseline, MFI = 12.26 +/- 1.77, p < 0.05), a functional feature distinct from IL-17 (MFI = 11.07 +/- 1.22). This effect was not inhibited by a saturating amount of IL-17. These findings demonstrate that ML-1 is a novel cytokine with a distinct function, and suggest a different receptor for ML-1 on PBE cells.     

HIF-1 activation attenuates postischemic myocardial injury: role for heme oxygenase-1 in modulating microvascular chemokine generation

The CXC chemokine IL-8, which promotes adhesion, activation, and transmigration of polymorphonuclear neutrophils (PMN), has been associated with production of tissue injury in reperfused myocardium. Hypoxia-inducible factor-1 (HIF-1) is a heterodimeric peptide that is a key regulator of genes such as heme oxygenase (HO)-1 expressed under hypoxic conditions. We hypothesized that HO-1 plays an important role in regulating proinflammatory mediator production under conditions of ischemia-reperfusion. HIF-1 was activated in the human microvascular endothelial cell line (HMEC-1) with the prolyl hydroxylase inhibitor dimethyloxalylglycine (DMOG). DMOG significantly attenuated cytokine-induced IL-8 promoter activity and protein secretion and cytokine-induced PMN migration across human microvascular endothelial cell line HMEC-1 monolayers. In vivo studies in a rabbit model of myocardial ischemia-reperfusion showed that rabbits pretreated with a 20 mg/kg DMOG infusion (n = 6) 24 h before study exhibited a 21.58 +/- 1.76% infarct size compared with 35.25 +/- 2.06% in saline-treated ischemia-reperfusion animals (n = 6, change in reduction = 39%; P < 0.001). In DMOG-pretreated (20 mg/kg) animals, plasma IL-8 levels at 3 h after onset of reperfusion were 405 +/- 40 pg/ml vs. 790 +/- 40 pg/ml in saline-treated ischemia-reperfusion animals (P < 0.001). DMOG pretreatment reduced myocardial myeloperoxidase activity, expressed as number of PMN per gram of myocardium, to 1.43 +/- 0.59 vs. 4.86 +/- 1.1 (P = 0.012) in saline-treated ischemia-reperfused hearts. Both in vitro and in vivo DMOG-attenuated IL-8 production was associated with robust HO-1 expression. Thus our data show that HIF-1 activation induces substantial HO-1 expression that is associated with attenuated proinflammatory chemokine production by microvascular endothelium in vitro and in vivo.     

Plasma adrenomedullin and endothelin-1 concentration during low-dose dobutamine infusion: Relationship between pulmonary uptake and pulmonary vascular pressure/flow characteristics

AIM: To study the role of endothelin (ET-1) and adrenomedullin (AM) on pulmonary vascular pressure/flow characteristic (pulmonary arterial pressure/cardiac output (Pap/CO)) during low-dose dobutamine infusion. METHODS: Case control study of 14 patients (12 men, 2 women) with severe lung disease (chronic obstructive pulmonary disease, COPD n=5; cystic fibrosis, CF n=9) and 5 control subjects (CTRL, 4 men, 1 woman). ET-1 and AM plasma levels in pulmonary artery (mixed venous blood, ven) and aorta or femoral artery (arterial, art), were measured at baseline and during dobutamine infusion (5-10-15 mcg kg(-1) min(-1)). The Ppa/CO coordinates obtained at baseline and during dobutamina infusion for each patients were used to calculate the Slope and Intercept by linear regression analysis. RESULTS: Baseline hemodynamics measurements were similar in the three groups with a trend towards a mild elevation in Ppa in CF group (Ppa mm Hg: CTRL 19+/-3.5, COPD 19.4+/-5.5, CF 22.7+/-7.5). Baseline plasma ET-1(ET-1ven pg ml(-1): CTRL 13.9+/-6.7, COPD 20.1+/-14, CF 20.4+/-7.1; ET-1art pg ml(-1): CTRL 16.7+/-6.4, COPD 20.1+/-11.7, CF 18.1+/-3.9) and AM (AMven pg ml(-1): CTRL 15.8+/-5, COPD 31.8+/-17.6, CF 27.7+/-7.6; AMart pg ml(-1): CTRL 15.9+/-1.4, COPD 21.4+/-3.8, CF 27+/-7.6) showed a trend towards higher value among patients' groups compared to the controls. Baseline ET-1 pulmonary gradient did not show significant difference among the three groups as well AM pulmonary gradient. Dobutamine infusion caused a comparable increase of heart rate and CO in the three groups. Mean pulmonary pressure had a trend towards a greater increase in COPD and CF than in controls, consequently, pulmonary Pap/CO relationship showed a steeper slope in patients' groups (Slope mm Hg L(-1) min(-1): CTRL 0.9+/-0.3, COPD 2.1+/-0.8 p<0.02 vs. CTRL, CF 1.9+/-0.9 p<0.03 vs CTRL). During dobutamine plasma ET-1 and AM showed a great individual variability resulting in no significant difference among groups. ET-1 pulmonary gradient showed a trend towards pulmonary uptake in patients' groups (ET-1art-ven pg min(-1): CTRL 2.7+/-2.9, COPD-6.1+/-7.8, CF -4+/-4.8) while AM pulmonary gradient did not show any particular pattern. During dobutamine ET-1 was significantly correlated to Pap/CO characteristics (Slope and ET-1ven, r=-0.59, p<0.05; Slope and ET-1art-ven, r=-0.60, p<0.05; Intercept and ET-1art-ven, r=0.63, p<0.004), and ET-1art-ven was the only independent variable related to Slope and Intercept. CONCLUSIONS: In patients with moderate pulmonary vascular impairment, ET-1 pulmonary gradient, but not AM pulmonary gradient, is inversely correlated with pulmonary incremental resistance, suggesting a role of ET-1 in the regulation of pulmonary vascular resistance.     

Differential regulation of cytokine and chemokine production in lipopolysaccharide-induced tolerance and priming

LPS pretreatment of human pro-monocytic THP-1 cells induces tolerance to secondary LPS stimulation with reduced TNFalpha production. However, secondary stimulation with heat-killed Staphylococcus aureus (HKSa) induces priming as evidenced by augmented TNFalpha production. The pro-inflammatory cytokine, IFNgamma, also abolishes suppression of TNFalpha in LPS tolerance. The effect of LPS tolerance on HKSa and IFNgamma-induced inflammatory mediator production is not well defined. We hypothesized that LPS, HKSa and IFNgamma differentially regulate pro-inflammatory mediators and chemokine production in LPS-induced tolerance. THP-1 cells were pretreated for 24 h with LPS (100 ng/ml) or LPS (100 ng/ml) + IFNgamma (1 microg/ml). Cells were subsequently stimulated with LPS or HKSa (10 microg/ml) for 24 h. The production of the cytokines TNFalpha, IL-6, IL-1beta, and GMCSF and the chemokine IL-8 were measured in supernatants. LPS and HKSa stimulated TNFalpha (3070 +/- 711 pg/ml and 217 +/- 9 pg/ml, respectively) and IL-6 (237 +/- 8.9 pg/ml and 56.2 +/- 2.9 pg/ml, p < 0.05, n = 3, respectively) in control cells compared to basal levels (< 25 pg/ml). LPS induced tolerance to secondary LPS stimulation as evidenced by a 90% (p < 0.05, n = 3) reduction in TNFalpha. However, LPS pretreatment induced priming to HKSa as demonstrated by increased TNFalpha (2.7 fold, from 217 to 580 pg/ml, p < 0.05, n = 3 ). In contrast to suppressed TNFalpha, IL-6 production was augmented to secondary LPS stimulation (9 fold, from 237 to 2076 pg/ml, p < 0.01, n = 3) and also primed to HKSa stimulation (62 fold, from 56 to 3470 pg/ml, p < 0.01, n = 3). LPS induced IL-8 production and to a lesser extent IL-1beta and GMCSF. LPS pretreatment did not affect secondary LPS stimulated IL-8 or IL-1beta, although HKSa stimulation augmented both mediators. In addition, IFNgamma pretreatment reversed LPS tolerance as evidenced by increased TNFalpha levels while IL-6, IL-1beta, and GMCSF levels were further augmented. However, IL-8 production was not affected by IFNgamma. These data support our hypothesis of differential regulation of cytokines and chemokines in gram-negative- and gram-positive-induced inflammatory events. Such changes may have implications in the pathogenesis of polymicrobial sepsis.     

Fluid shear stress suppresses interleukin 8 production by vascular endothelial cells.

The effects of shear stress on interleukin 8 (IL-8) production by human umbilical vein endothelial cells (HUVEC) were studied by subjecting the HUVEC to a steady flow laminar shear stress of up to 0.7 N/m(2) in a parallel plate flow chamber. Shear stress decreased IL-8 mRNA expression in a dose and time-dependent fashion. High glucose concentrations increased IL-8 mRNA levels in a MAPK-p38-dependent manner, which was suppressed by shear stress. Measurement of IL-8 protein in HUVEC culture media by ELISA demonstrated that IL-8 secretion was also increased by high glucose and suppressed by shear stress. These results suggest that the anti-atherogenic effect of shear stress arises partly from the suppression of the production of IL-8 which has been shown to trigger the adhesion of monocytes to a vascular endothelium and also acts as a mitogen and chemoattractant for vascular smooth muscle cells.     

Elevated levels of interleukin-13 and IL-18 in patients with dengue hemorrhagic fever

Interleukin (IL)-13 is produced by T helper 2 (Th2)-type cells and inhibits the production of proinflammatory cytokines by activated monocytes, while IL-18 is a pleiotropic cytokine that induces interferon-gamma and plays an important role in the development of Th1-type cells. Role of the shift from a Th1-type response to Th2-type has been suggested in the pathogenesis of dengue hemorrhagic fever (DHF). This study was undertaken to investigate the possible protective/pathogenic role of IL-13 and IL-18 in patients with DHF. Sera were collected from a total of 84 patients with various grades of dengue illness and 21 normal healthy controls and tested for IL-13 and IL-18 levels using commercial enzyme-linked immunosorbent assay kits. The results showed that very low levels of IL-13 (4+/-3 pg ml(-1)) and IL-18 (15+/-4 pg ml(-1)) were detected in the sera of healthy controls. In dengue patients, the levels of IL-13 and IL-18 were the highest in the patients with DHF grade IV (205+/-103 pg ml(-1) and 366+/-155 pg ml(-1), respectively) and the lowest in patients with dengue fever (22+/-12 pg ml(-1) and 76+/-50 pg ml(-1), respectively). Both the cytokines appeared (IL-13=20+/-11 pg ml(-1) and IL-18=70+/-45 pg ml(-1)) during the first 4 days of illness and reached peak levels (IL-13=204+/-96 pg ml(-1) and IL-18=360+/-148 pg ml(-1)) by day 9 onwards. The presence of high levels of IL-13 and IL-18 during severe illness and late phases of the disease suggests that both of these cytokines may contribute to the shift from a Th1- to Th2-type response and thus to the pathogenesis of DHF.     

Serum levels of TNF-alpha,sIL-2R,IL-6, and IL-8 are increased and associated with elevated lipid peroxidation in patients with Behçet's disease

AIM: Behçet''s disease (BD) is asystemic immunoinflammatory disorder and the aetiopathogenesis is to be specified. Cytokines play a role in immune response and in many inflammatory diseases. The aim of this case-control study is to investigate serum pro-inflammatory cytokine tumour necrosis factor (TNF)-alpha, interleukin-1beta (IL-1beta), soluble IL-2 receptor (sIL-2R), IL-6, and chemokine IL-8 levels in patients with BD. We also determined the end product of lipid peroxidation (malondialdehyde (MDA)) in BD patients as an index for oxidative stress. METHODS: A total of 37 patients (19 men, 18 women) with BD (active, n = 17; inactive, n = 20) and 20 age-matched and sex-matched healthy control subjects (11 men, nine women) included in this cross-sectional, blinded study. Serum TNF-alpha, IL-1beta, sIL-2R, IL-6 and IL-8 levels were determined by a spectrophotometer technique using the immulite chemiluminescent immunometric assay. Lipid peroxidation was evaluated by Wasowicz et aL The levels of cytokines and lipid peroxidation in the active period were compared with the inactive period of the disease. Results are expressed as mean +/- standard error. RESULTS: IL-1beta levels were below the detection limits of the assay (< 5 pg/ml) in all samples. Mean levels of MDA (8.1+/-0.7 micromol/l), sIL-2R (800+/-38 U/ml), IL-6 (12.6+/-1.1 pg/ml), IL-8 (7.2+/-0.4 pg/ml), and TNF-alpha (7.9+/-0.5 pg/ml) in active BD patients were significantly higher than those in inactive patients (4.3+/-0.5 micromol/l, p < 0.01; 447+/-16 U/ml, p < 0.001; 8.3+/-0.6 pg/ml, p = 0.006; 5.3+/-0.1 pg/ml, p < 0.001; and 5.1 0.2 pg/ml, p < 0.001; respectively) or control subjects (2.1+/-0.2 micromol/l, p < 0.001; 446+/-20 U/ml, p < 0.001; 6.4+/-0.2 pg/ml, p < 0.001; 5.4+/-0.1 pg/ml, p < 0.001; and 4.7+/-0.1 pg/ml, p < 0.001, respectively). On the contrary, only the mean IL-6 level was significantly different between inactive BD and control subjects (p = 0.02). All acute phase reactants were significantly higher in active BD than in inactive period (for each, p < 0.01). Conclusions: High levels of sIL-2R, IL-6, IL-8 and TNF-alpha indicate the activation of immune system in BD. Serum sIL-2R, IL-6, IL-8 and TNF-alpha seem to be related to disease activity. Increased lipid peroxidation suggests oxidative stress in BD and therefore tissue damage in such patients. Amelioration of clinical manifestations would be envisaged by targeting these cytokines, chemokines and lipid peroxidation with pharmacological agents.