Search

Introduction. Chronic pulmonary infection is associated with colonization with multiple micro-organisms but host-microbe and microbe-microbe interactions are poorly understood.Aim. This study aims to investigate the differences in host responses to mono- and co-infection with S. aureus and B. cenocepacia in human airway epithelial cells.Methodology. We assessed the effect of co-infection with B. cenocepacia and S. aureus on host signalling and inflammatory responses in the human airway epithelial cell line 16HBE, using ELISA and western blot analysis.Results. The results show that B. cenocepacia activates MAPK and NF-κB signalling pathways, subsequently eliciting robust interleukin (IL)-8 production. However, when airway epithelial cells were co-treated with live B. cenocepacia bacteria and S. aureus supernatants (conditioned medium), the pro-inflammatory response was attenuated. This anti-inflammatory effect was widely exhibited in the S. aureus isolates tested and was mediated via reduced MAPK and NF-κB signalling, but not via IL-1 receptor or tumour necrosis factor receptor modulation. The staphylococcal effectors were characterized as small, heat-stable, non-proteinaceous and not cell wall-related factors.Conclusion. This study demonstrates for the first time the host response in a S. aureus/B. cenocepacia co-infection model and provides insight into a staphylococcal immune evasion mechanism, as well as a therapeutic intervention for excessive inflammation.

Background Advanced glycation endproducts (AGEs) play a major role in the development of many vascular complications that are mediated by endothelial dysfunction. The present work aimed to investigate the mechanism by which AGEs impair vasodilation. Methods The effect of AGEs on vasodilation induced by acetylcholine or D NONOate was examined by incubating isolated rat aortae with different AGEs concentrations. ACh-induced nitric oxide generation was assessed using the fluorescent probe diaminofluorecein (DAF-FM). The effect of AGEs on expression of mRNA for arginase 2, NADPH oxidase and endothelial nitric oxide synthase (eNOS) were determined by real-time PCR. Results One-hour in vitro incubation of rat aortae with AGEs impaired endothelial-dependent vasodilation produced by ACh, while increasing D NONOate-induced vasodilation. Preincubation of aortae with l -ornithine, an arginase 2-inhibitor, prevented the impairment effect induced by AGEs on endothelial-dependent vasodilation. Superoxide scavenging by tempol or NADPH oxidase inhibition by apocynin also blocked the effect of AGEs. AGEs decreased ACh-induced NO production and this was inhibited by both l -ornithine and apocynin. Furthermore, AGEs exposure increased arginase mRNA expression but decreased mRNA expression for eNOS in isolated rat aortae. Conclusion The present results indicate that AGEs impairs endothelial-dependent vasodilation, and this effect is mediated via arginase overexpression and NADPH oxidase stimulation.

Salah A Ghareib,1 Hany M El-Bassossy,1,2 Ahmed A Elberry,3,4 Ahmad Azhar,5 Malcolm L Watson,6 Zainy Mohammed Banjar7 1Department of Pharmacology and Toxicology, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia; 2Department of Pharmacology, Faculty of Pharmacy, Zagazig University, Zagazig, Egypt; 3Department of Clinical Pharmacy, Faculty of Pharmacy, King Abdulaziz University, Jeddah, Saudi Arabia; 4Department of Pharmacology, Faculty of Medicine, Beni Suef University, Beni Suef, Egypt; 5Department of Pediatric Cardiology, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia; 6Department of Pharmacy and Pharmacology, University of Bath, Bath, UK; 7Department of Clinical Biochemistry, Faculty of Medicine, King Abdulaziz University, Jeddah, Saudi Arabia Abstract: The aim of the present study is to investigate the effect and potential mechanism of action of 6-gingerol on alterations of vascular reactivity in the isolated aorta from diabetic rats. Male Wistar rats were divided into two experimental groups, control and diabetics. Diabetes was induced by a single intraperitoneal injection of streptozotocin (50mg kg-1), and the rats were left for 10weeks to develop vascular complications. The effect of in vitro incubationwith 6-gingerol (0.3–3µM) on the vasoconstrictor response of the isolated diabetic aortae to phenylephrine and the vasodilator response to acetylcholine was examined. Effect of 6-gingerol was also examined on aortae incubated with methylglyoxal as an advanced glycation end product (AGE). To investigate the mechanism of action of 6-gingerol, the nitric oxide synthase inhibitor Nω-nitro-L-arginine methyl ester hydrochloride (100µM), guanylate cyclase inhibitor methylene blue (5µM), calcium-activated potassium channel blocker tetraethylammonium chloride (10mM), and cyclooxygenase inhibitor indomethacin (5µM) were added 30minutes before assessing the direct vasorelaxant effect of 6-gingerol. Moreover, in vitro effects of 6-gingerol on NO release and the effect of 6-gingerol on AGE production were examined. Results showed that incubation of aortae with 6-gingerol (0.3–10µM) alleviated the exaggerated vasoconstriction of diabetic aortae to phenylephrine in a concentration-dependent manner with no significant effect on the impaired relaxatory response to acetylcholine. Similar results were seen in the aortae exposed to methylglyoxal. In addition, 6-gingerol induced a direct vasodilation effect that was significantly inhibited by Nω-nitro-L-arginine methyl ester hydrochloride and methylene blue. Furthermore, 6-gingerol stimulated aortic NO generation but had no effect on AGE formation. In conclusion, 6-gingerol ameliorates enhanced vascular contraction in diabetic aortae, which may be partially attributed to its ability to increase the production of NO and stimulation of cyclic guanosine monophosphate. Keywords: diabetes, 6-gingerol, vasorelaxant, nitric oxide, advanced glycation end products, vascular complications

Background and Purpose We have previously shown that arginase inhibition alleviates hypertension associated with in a diabetic animal model. Here, we investigated the protective effect of arginase inhibition on hypertension in metabolic syndrome. Experimental Approach Metabolic syndrome was induced in rats by administration of fructose (10% in drinking water) for 12 weeks to induce vascular dysfunction. Three arginase inhibitors (citrulline, norvaline and ornithine) were administered daily in the last 6 weeks of study before and tail BP was recorded in conscious animals. Concentration response curves for phenylephrine (PE), KCl and ACh in addition to ACh-induced NO generation were obtained in thoracic aorta rings. Serum glucose, insulin, uric acid and lipid profile were determined as well as reactive oxygen species (ROS) and arginase activity. Key Results Arginase activity was elevated in metabolic syndrome while significantly inhibited by citrulline, norvaline or ornithine treatment. Metabolic syndrome was associated with elevations in systolic and diastolic BP, while arginase inhibition significantly reduced elevations in diastolic and systolic BP. Metabolic syndrome increased vasoconstriction responses of aorta to PE and KCl and decreased vasorelaxation to ACh, while arginase inhibition completely prevented impaired responses to ACh. In addition, arginase inhibition prevented impaired NO generation and exaggerated ROS formation in metabolic syndrome. Furthermore, arginase inhibition significantly reduced hyperinsulinaemia and hypertriglyceridaemia without affecting hyperuricaemia or hypercholesterolaemia associated with metabolic syndrome. Conclusions and Implications Arginase inhibition alleviates hypertension in metabolic syndrome directly through endothelial-dependent relaxation/NO signalling protection and indirectly through inhibition of insulin resistance and hypertriglyceridaemia.

The current study was conducted to evaluate the effect of 6-gingerol (6G) on cardiac complications in streptozotocin (STZ)-induced diabetic (DM) rats. STZ-induced DM rats (single 50 mg/kg i.p. injection, 15 days prior to drug treatment) or time-matched controls were treated with 6G (75 mg/day route orally). After a further 8 weeks, blood was collected for biochemical analysis and 8-isoprostenol was measured in urine. Cardiac hemodynamics and ECG was assessed. 6G significantly attenuated the increased level of blood glucose in diabetic rats and improved cardiac hemodynamics in including RR interval, max dP/dt, min dP/dt and Tau. In addition, 6G alleviated the elevated ST segment, T amplitude and R amplitude with no significant effect on disturbed levels of adiponectin, TGF-β or 8-isoprostenol induced by diabetes. The results showed that treatment with 6G has an ameliorative effect on cardiac dysfunction induced by diabetes. Which may be not related to its potential antioxidant effect.

The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 are involved in variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. Two alternatively spliced variants of the human CXCR3-A receptor have been described, termed CXCR3-B and CXCR3-alt. Human CXCR3-B binds CXCL9, CXCL10, CXCL11 as well as an additional ligand CXCL4. In contrast, CXCR3-alt only binds CXCL11. We report that CXCL4 induces intracellular calcium mobilization as well as Akt and p44/p42 extracellular signal-regulated kinase phosphorylation, in activated human T lymphocytes. These responses have similar concentration dependence and time-courses to those induced by established CXCR3 agonists. Moreover, phosphorylation of Akt and p44/p42 is inhibited by pertussis toxin, suggesting coupling to Gα(i) protein. Surprisingly, and in contrast with the other CXCR3 agonists, stimulation of T lymphocytes with CXCL4 failed to elicit migratory responses and did not lead to loss of surface CXCR3 expression. Taken together, our findings show that, although CXCL4 is coupled to downstream biochemical machinery, its role in T cells is probably distinct from that of CXCR3-A agonists.

Background and purpose: Disturbances in pulmonary vascular reactivity are important components of inflammatory lung disease. Haem oxygenase-1 (HO-1) is an important homeostatic enzyme upregulated in inflammation. Here we have investigated the potentially protective effect of HO-1 against cytokine-induced impairment in pulmonary artery relaxation. Experimental approach: Haem oxygenase-1 protein levels were assessed by immunofluorescence. HO activity was assessed by conversion of haemin to bilirubin. Rings of rat isolated pulmonary artery in organ baths were used to measure relaxant responses to the endothelium-dependent agent ACh and the endothelium-independent agent sodium nitroprusside (SNP). Production of nitric oxide (NO) and reactive oxygen species (ROS) was assessed by confocal fluorescence microscopy and fluorescent probes. Key results: Haem oxygenase-1 protein expression was strongly induced in pulmonary artery after 24-h incubation with either haemin (5 µM) or curcumin (2 µM), accompanied by a significant increase in HO activity. Incubation with tumour necrosis factor α (TNFα, 1 ng·mL−1, 2 h) significantly decreased relaxation of arterial rings to ACh, without affecting responses to SNP. Induction of HO-1 by curcumin or haemin protected against TNFα-induced hyporesponsiveness to ACh. The competitive HO inhibitor, tin protoporphyrin (20 µM), abolished the protective effect of haemin. HO-1 induction prevented a TNFα-induced increase in NO generation without affecting the TNFα-induced increase in ROS generation. HO-1 induction prevented the TNFα-induced decrease in ACh-stimulated NO generation. Conclusions and implications: Induction of HO-1 protected against TNFα impairment of endothelium-dependent relaxation in pulmonary artery, by a mechanism involving a reduction in inducible NO synthase-derived NO production.

The roles of intracellular calcium concentration ([Ca2+]i) and Ca2+sensitization in lipopolysaccharide (LPS)-induced vascular smooth muscle (VSM) hyporesponsiveness are incompletely understood. To investigate these roles, contraction responses to endothelin-1 (ET-1) and 80 mM KCl; relaxation responses to nifedipine; the expression levels of mRNAs of ET-1 and its receptors (ETAor ETB); the expression levels of protein kinase C (PKC) and phosphorylation of Rho kinase (ROKα), CPI-17, and myosin phosphatase target subunit-1 (MYPT1); and changes in aortic VSM cell [Ca2+]iwere measured in LPS-treated aortic rings from male Wistar rats (250–300 g). LPS (10 μg/ml, 20 h) decreased contraction induced by ET-1 (0.3–100 nM) or 80 mM KCl. LPS-induced hypocontractility was not observed in the absence of external Ca2+, but LPS-treated aorta remained hypocontractile on subsequent stepwise restoration of extracellular Ca2+(0.01–10 mM). Vascular relaxation to nifedipine; mRNA expression levels of ET-1, ETA, or ETB; protein expression levels of PKC; and phosphorylation levels of ROKα, CPI-17, and MYPT1 were not affected by LPS. In isolated aortic VSM cells, ET-1 caused a transient initial increase in [Ca2+]i, followed by a maintained tonic increase in [Ca2+]i, which was decreased by LPS pretreatment and was dependent on external Ca2+. Subsequent restoration of extracellular Ca2+increased [Ca2+]i, but this increase was lower in the LPS-treated group. This difference in response to extracellular Ca2+addition was not affected by diltiazem, but was abolished by SKF-96365. Therefore, LPS induces hyporeactivity to ET-1 in rat aorta that depends on external Ca2+influx through non-voltage-operated Ca2+channels, but not on ET-1 receptor expression or Ca2+sensitization.

Background and purpose: To investigate the function of soluble guanylyl cyclase (sGC)/3′,5′-cyclic guanosine monophosphate (cGMP) pathway in lipopolysaccharide (LPS)-induced changes in vascular reactivity of rat isolated pulmonary artery and aorta. Experimental approach: Nitric oxide (NO) production, contraction responses to endothelin-1 (ET-1), relaxation responses to sodium nitroprusside (SNP), 8-pCPT-cGMP, BAY412272 and T-0156, SNP-induced cGMP production and expression of sGCα1, sGCβ1 and 3′,5′-cyclic nucleotide phosphodiesterase-5 (PDE5) proteins were measured in LPS-treated pulmonary and aortic rings from male Wistar rats. Key results: In both vessels, LPS (10 μg mL−1, 20 h) increased NO production, which was inhibited by the selective inducible NOS (iNOS) inhibitor 1400W (1 μM). In the aorta, LPS decreased ET-1-induced contractility and this decrease was inhibited by the selective sGC inhibitor ODQ (10 μM) but not by removal of endothelium, or inhibitors of cyclooxygenase (indomethacin, 10 μM) or iNOS (1400W, 1 μM). Furthermore, aortic relaxation responses to the direct sGC activator BAY412272 were enhanced. In the pulmonary artery, SNP (1 nM to 30 μM)-induced relaxation and cGMP production, BAY412272-induced relaxation and sGCβ1 protein expression were decreased, whereas relaxation responses to the PDE5-specific inhibitor T-0156 (0.1–100 nM) were enhanced. Relaxation responses to the phosphodiesterase-resistant cGMP analogue, 8-pCPT-cGMP, and protein expression levels of sGCα1 and PDE5 were not altered in either vessel. Conclusion and implications: LPS caused a selective hypocontractility of rat aorta to ET-1 mediated mainly through NO-independent sGC activation, whereas in the pulmonary artery, the effect of sGC activation was reduced by a decreased protein expression of sGCβ1 together with increased PDE5 activity. British Journal of Pharmacology (2008) 155, 1164–1173; doi:10.1038/bjp.2008.358; published online 22 September 2008

BackgroundWe have previously shown that hyperuricemia plays an important role in the vascular complications of insulin resistance (IR). Here we investigated the effect of xanthine oxidase (XO) inhibition on the cardiac complications of IR. MethodsIR was induced in rats by a high fructose high fat diet for 12 weeks. Allopurinol, a standard XO inhibitor, was administered in the last 4 weeks before cardiac hemodynamics and electrocardiography, serum glucose, insulin, tumor necrosis factor alpha (TNFα), 8-isoprostane, uric acid, lactate dehydrogenase (LDH) and XO activity were measured. Expression of cardiac angiotensin II (AngII) and angiotensin receptor 1 (AT1) were assessed by immunofluorescence. ResultsIR animals had significant hyperuricemia which was inhibited by allopurinol administration. IR was associated with impaired ventricular relaxation (reflected by a decreased diastolic pressure increment and prolonged diastolic duration) and XO inhibition greatly attenuated impaired relaxation. IR was accompanied by cardiac ischemia (reflected by increased QTc and T peak trend intervals) while XO inhibition alleviated the ECG abnormalities. When subjected to isoproterenol-induced ischemia, IR hearts were less resistant (reflected by larger ST height depression and higher LDH level) while XO inhibition alleviated the accompanying ischemia. In addition, XO inhibition prevented the elevation of serum 8-isoprostane and TNFα, and blocked elevated AngII and AT1 receptor expression in the heart tissue of IR animals. However, XO inhibition did not affect the developed hyperinsulinemia or dyslipidemia. ConclusionsXO inhibition alleviates cardiac ischemia and impaired relaxation in IR through the inhibition of low grade inflammation and the angiotensin system.

The Th2 cytokine interleukin (IL)-13 is believed to play an important role in the development of allergy, although it has also been ascribed anti-inflammatory roles in several experimental models. In this study, we have examined the effects of human recombinant IL-13 on eosinophilic lung inflammation in the guinea pig. IL-13 (1 to 100 ng, given by intratracheal instillation) did not elicit airway eosinophil recruitment. A pronounced accumulation of eosinophils, as well as monocyte/macrophages, was elicited by intratracheal instillation of guinea pig tumor necrosis factor alpha (gpTNF-alpha). Intratracheal administration of IL-13 (1 to 100 ng) given immediately prior to exposure to gpTNF-alpha resulted in a dose-related suppression of eosinophil and monocyte/macrophage accumulation in the airways, as assessed by bronchoalveolar lavage (BAL) and eosinophil peroxidase activity in whole-lung homogenates. IL-13 treatment also reduced BAL fluid (BALF) leukocyte accumulation induced by subsequent aerosol antigen challenge of sensitized guinea pigs. Antigen challenge also resulted in elevated levels of immunoreactive eotaxin and eosinophil-stimulating activity in BALF, although only the latter was reduced significantly by IL-13 instillation prior to challenge. In contrast to the suppressive effects of IL-13, instillation of human recombinant IL-4 (100 ng) alone elicited an increase in BALF monocyte/macrophage numbers, and IL-4 was unable to inhibit gpTNF-alpha-induced leukocyte accumulation. Hence, IL-13 (but not human IL-4) exhibits an anti-inflammatory action in the airways of gpTNF-alpha- or antigen-challenged guinea pigs, by mechanisms that may involve the decreased generation of eosinophil-stimulating activity in the airways.

1. The production of chemokines by vascular smooth muscle cells (SMC) is implicated in the pathogenesis of atherosclerosis, although the factors regulating chemokine production by these cells are incompletely characterized. 2. We describe the differential stimulation of interleukin-(IL)-8, monocyte chemoattractant protein (MCP)-1 and regulated on activation normal T-cell expressed and secreted (RANTES) synthesis following treatment of human vascular SMC with IL-1alpha or tumour necrosis factor alpha (TNFalpha). Under basal conditions, cultured SMC release very low amounts of IL-8, MCP-1 and RANTES as assessed by specific ELISA. Concentration-response studies with IL-1alpha or TNFalpha revealed that each stimulus induced a similar amount of MCP-1. In contrast approximately three fold more IL-8 was induced by IL-1alpha than by TNFalpha whereas significant RANTES production was induced only by TNFalpha. These findings point to a divergence in the regulation of synthesis of the different chemokines in response to IL-1alpha or TNFalpha stimulation. 3. The T-cell derived cytokines IL-10 and IL-13 were also found to have differential effects on chemokine production by SMC. IL-13, but not IL-10, significantly enhanced IL-8 and MCP-1 release in response to IL-1alpha or TNFalpha. This increase in chemokine release appeared to be accounted for by increased mRNA expression. 4. These findings provide support for the concept that smooth muscle cells can have an active role in a local immune response via the production of chemokines which can be selectively modulated by T-cell derived cytokines.

We have cloned and expressed recombinant guinea pig tumor necrosis factor-alpha (gpTNF-alpha) and examined its inflammatory activities after tracheal instillation in guinea pigs. A 1,071-bp cDNA, including the region encoding the full-length 234-amino acid gpTNF-alpha protein, was cloned from concanavalin A-stimulated guinea pig splenocytes. The 154-amino acid protein corresponding to secreted gpTNF-alpha was expressed as a fusion protein in Escherichia coli, purified by affinity chromatography, and cleaved to yield a 17-kDa protein. gpTNF-alpha had a cytotoxic effect on WEHI 164 cells and was detected by goat anti-murine tumor necrosis factor-alpha (TNF-alpha) antibody in Western blots. Intratracheal instillation of gpTNF-alpha (50-150 ng) caused pronounced and dose-dependent airway eosinophilia. Incubation of gpTNF-alpha with rabbit anti-murine TNF-alpha sera or heating the gpTNF-alpha before instillation reduced bronchoalveolar lavage (BAL) eosinophils to near control levels. Maximum BAL eosinophilia was observed at 24 h, but eosinophil numbers remained significantly above vehicle-treated animals for 72 h. Hence, gpTNF-alpha elicits a pronounced and protracted eosinophil accumulation in the guinea pig lung.

Cultured human synovial fibroblasts express mRNA for the chemotactic cytokines (chemokines) interleukin-8 (IL-8), monocyte chemotactic protein 1 (MCP-1) and regulated upon activation normal T-cell expressed and presumably secreted (RANTES), when stimulated with IL-1 or tumour necrosis factor alpha (TNF alpha). Calyculin A, a potent type 1/2A protein serine/threonine phosphatase inhibitor, was used to examine the role of protein phosphatases in the regulation of chemokine gene expression. Calyculin A (1 nM) mimicked IL-1 by inducing IL-8 and MCP-1 mRNA expression in synovial cells. IL-8 mRNA was induced over a similar time period (1-6 h) in response to IL-1 or calyculin A, whereas MCP-1 mRNA was induced more rapidly (1-2 h) by calyculin A than by IL-1 (4-6 h). Expression of RANTES mRNA occurred in response to TNF alpha, but could not be induced by stimulation with calyculin A alone. These results suggest that inhibition of protein phosphatase type 1/2A may have a differential role in the regulation of the expression of each of the chemokine genes. Synovial fibroblasts also secreted IL-8 and IL-6 peptide when stimulated with either IL-1/TNF alpha or calyculin A. The amount of IL-8 and IL-6 peptide produced in response to calyculin A was significantly increased above that produced by untreated synovial cells, though it was much less than the amount induced by IL-1 or TNF alpha. Calyculin A also acted synergistically with IL-1 or TNF alpha to cause a 2-fold potentiation of IL-1- or TNF alpha-induced IL-8 mRNA and peptide and RANTES mRNA expression. These results suggest that although inhibition of a protein phosphatase may be able to regulate the magnitude of IL-1-induced chemokine gene expression, the IL-1 signal transduction pathway involves components in addition to phosphatase inhibition, possibly including the activation of a protein kinase, the action of which may be opposed by a protein phosphatase inhibited by calyculin A.

1. Eosinophil accumulation and plasma extravasation are features of type I allergic responses. In an attempt to characterize the mediators of these responses, we have examined the local accumulation of 111In-eosinophils and leakage of 125I-human serum albumin (125I-HSA) during passive cutaneous anaphylaxis (PCA) reactions and in response to defined inflammatory mediators in the guinea-pig. Animals were passively sensitized by intradermal injection of anti-bovine gamma globulin antibody (50 microliters, 1/50 dilution). After 20-24 h, animals were injected intravenously with 111In-eosinophils and 125I-HSA for the measurement of cell accumulation and plasma leakage, respectively. 2. When injected into sensitized sites, antigen caused a dose-related increase in the accumulation of 111In-eosinophils and plasma leakage in guinea-pig skin. Time course experiments over 24 h revealed that the maximal rate of 111In-eosinophil accumulation occurred over the first 90 min, with little accumulation at later time points. Plasma leakage was completed within the first 30 min after challenge. Responses to the mast cell degranulator, compound 48/80, exhibited very similar responses to the PCA reaction. 3. Co-injection of antigen with the PAF antagonist, WEB 2086 (10(-7) mol/site) or the 5-lipoxygenase inhibitor, PF 5901 (10(-7) mol/site) did not significantly alter the accumulation of 111In-eosinophils or plasma leakage, whereas these drug doses abolished responses to exogenous PAF (10(-9) mol/site) and arachidonic acid (AA, 3 x 10(-8) mol/site), respectively. The H1 receptor antagonist chlorpheniramine (2.5 x 10(-8) mol/site) did not reduce antigen-induced 111In-eosinophil accumulation. Drug combinations were also injected with antigen into sensitized sites, but were unable to reduce "'In-eosinophil accumulation.4. These results indicate that anaphylactic eosinophil accumulation in this model involves mediators other than histamine, PAF or lipoxygenase products. This is in contrast to plasma leakage in this reaction, which can be abolished by a combination of antagonists blocking these mediators.

Objectives Laser speckle contrast imaging (LSCI) is a novel non-invasive microvascular imaging modality. The present study evaluates the validity and reliability of LSCI by comparison with infrared thermography (IRT) for the dynamic assessment of digital microvascular function in healthy volunteers. Methods Subjects attended on 3 occasions. Simultaneous assessment of cutaneous perfusion at 3 distinct regions of interest (ROI) within the hands was undertaken using LSCI and infrared thermography (IRT) at baseline, and at 13 s intervals over 15 min following a standardised local cold challenge. Endpoints for evaluation included absolute measurements at baseline and following cold stress, in addition to the characteristics of the re-warming curves (maximum % recovery and maximum gradient). Visits 1 and 2 were undertaken in identical conditions (ambient temperature 23 °C) to assess reproducibility, whereas visit 3 was undertaken at a lower ambient room temperature of 18 °C to evaluate responsiveness to reduction in ambient room temperature. Results Fourteen healthy participants completed the study. There was greater variability in the data generated using LSCI compared with the highly damped IRT, reflecting greater sensitivity of LSCI to physiological variation and movement artefact. LSCI and IRT correlated well at baseline and following cold challenge for all endpoints (rs for pooled data between 0.5 and 0.65, p 0.75) for absolute assessments but lower for re-warming curve characteristics. LSCI provides greater spatial resolution than IRT identifying variation in cutaneous perfusion within the hands most likely associated with the presence of arteriovenous anastamoses. Both techniques were responsive to reduction in ambient room temperature. Effect sizes were greatest for IRT than LSCI (e.g. − 1.17 vs. − 0.85 at ROI 1 at baseline) although this may represent heat transfer rather than altered vascular perfusion. Discussion In the dynamic assessment of digital vascular perfusion, LSCI correlates well with IRT, is reproducible and responsive to reduction in ambient room temperature. Absolute measurements appear preferable to parameters derived from re-warming curve characteristics when assessing digital perfusion following cold challenge. The greater temporal and spatial resolution of LSCI compared with IRT may facilitate the development of novel assessment tools of autonomic function and digital cutaneous perfusion.

The chemokine receptor CXCR3 and its ligands CXCL9, CXCL10 and CXCL11 are involved in variety of inflammatory disorders including multiple sclerosis, rheumatoid arthritis, psoriasis and sarcoidosis. Two alternatively spliced variants of the human CXCR3-A receptor have been described, termed CXCR3-B and CXCR3-alt. Human CXCR3-B binds CXCL9, CXCL10, CXCL11 as well as an additional ligand CXCL4. In contrast, CXCR3-alt only binds CXCL11. We report that CXCL4 induces intracellular calcium mobilization as well as Akt and p44/p42 extracellular signal-regulated kinase phosphorylation, in activated human T lymphocytes. These responses have similar concentration dependence and time-courses to those induced by established CXCR3 agonists. Moreover, phosphorylation of Akt and p44/p42 is inhibited by pertussis toxin, suggesting coupling to Gα(i) protein. Surprisingly, and in contrast with the other CXCR3 agonists, stimulation of T lymphocytes with CXCL4 failed to elicit migratory responses and did not lead to loss of surface CXCR3 expression. Taken together, our findings show that, although CXCL4 is coupled to downstream biochemical machinery, its role in T cells is probably distinct from that of CXCR3-A agonists.

The role of pro-inflammatory cytokines in an animal model of allergic lung disease was examined by use of an interleukin-1 receptor antagonist (IL-1ra) and a specific bioassay for tumor necrosis factor (TNF). Ovalbumin-sensitized guinea pigs exhibit a marked bronchial hyperreactivity (assessed by airway responsiveness to intravenous histamine) and pulmonary eosinophil accumulation (assessed by bronchoalveolar lavage) 24 h after challenge with aerosolized antigen. Exposure of animals to an aerosol of IL-1ra (50 micrograms over 30 min) immediately before antigen challenge resulted in a marked protection against bronchial hyperreactivity and pulmonary eosinophil accumulation compared with IL-1ra vehicle-pretreated animals. Additionally, we report for the first time generation of TNF bioactivity in the bronchoalveolar lavage of antigen-challenged animals, which was significantly reduced in animals exposed to aerosolized IL-1ra before challenge. These studies point to a key role for the cytokines IL-1 and possibly TNF in the pulmonary changes observed during allergic airway disease.

Members of the chemokine (Chemotactic cytokines) superfamily and their receptors play a major role in trafficking of immune cells under homeostatic and inflammatory conditions. The chemokine receptor CXCR3 is expressed mainly on activated T lymphocytes and binds three pro-inflammatory, interferon-gamma-inducible chemokines: monokine induced by IFN-gamma (Mig/CXCL9), IFN-gamma-induced protein-10 (IP-10/CXCL10) and IFN-gamma-inducible T-cell alpha-chemoattractant (I-TAC/CXCL11). CXCR3 and its agonists are involved in a variety of inflammatory pathologies, making this receptor an attractive target for the design of new anti-inflammatory drugs. Interestingly, a growing body of evidence suggests the existence of at least two novel variants of CXCR3, namely CXCR3-B and CXCR3-alt, which present challenges in the design of new anti-inflammatory drugs targeting CXCR3. In this chapter, we describe the collection, isolation and activation of human peripheral blood-derived T lymphocytes and methods to examine the expression of CXCR3 and its atypical variants at both mRNA and protein levels, as well as protocols for exploring the biochemical and functional responses of T lymphocytes to all known CXCR3 agonists.

Disturbances in pulmonary vascular reactivity are important components of inflammatory lung disease. Haem oxygenase-1 (HO-1) is an important homeostatic enzyme upregulated in inflammation. Here we have investigated the potentially protective effect of HO-1 against cytokine-induced impairment in pulmonary artery relaxation.Haem oxygenase-1 protein levels were assessed by immunofluorescence. HO activity was assessed by conversion of haemin to bilirubin. Rings of rat isolated pulmonary artery in organ baths were used to measure relaxant responses to the endothelium-dependent agent ACh and the endothelium-independent agent sodium nitroprusside (SNP). Production of nitric oxide (NO) and reactive oxygen species (ROS) was assessed by confocal fluorescence microscopy and fluorescent probes.Haem oxygenase-1 protein expression was strongly induced in pulmonary artery after 24-h incubation with either haemin (5 microM) or curcumin (2 microM), accompanied by a significant increase in HO activity. Incubation with tumour necrosis factor alpha (TNFalpha, 1 ng.mL(-1), 2 h) significantly decreased relaxation of arterial rings to ACh, without affecting responses to SNP. Induction of HO-1 by curcumin or haemin protected against TNFalpha-induced hyporesponsiveness to ACh. The competitive HO inhibitor, tin protoporphyrin (20 microM), abolished the protective effect of haemin. HO-1 induction prevented a TNFalpha-induced increase in NO generation without affecting the TNFalpha-induced increase in ROS generation. HO-1 induction prevented the TNFalpha-induced decrease in ACh-stimulated NO generation.Induction of HO-1 protected against TNFalpha impairment of endothelium-dependent relaxation in pulmonary artery, by a mechanism involving a reduction in inducible NO synthase-derived NO production.

The family of lipid kinases termed phosphoinositide-3-kinase (PI3K) is known to contribute at multiple levels to innate and adaptive immune responses, and is hence an attractive target for drug discovery in inflammatory and autoimmune disease, including respiratory diseases. The development of isoform-selective pharmacological inhibitors, targeted gene manipulation and short interfering RNA (siRNA) target validation have facilitated a better understanding of the role that each member of this family of kinases plays in the physiology and pathology of the respiratory system. In this review, we will evaluate the evidence for the roles of specific PI3K isoforms in the lung and airways, and discuss their potential as targets for novel drug therapies.

Ligands of peroxisome proliferator-activated receptor-gamma (PPAR(gamma)) are thought to possess anti-inflammatory properties mediated via both PPAR(gamma) dependent and independent mechanisms. This work investigates the effects of PPAR(gamma) ligands on the regulation of cyclooxygenase-2 (COX-2) in the human lung epithelial cell line, A549. The synthetic ligand troglitazone activated the phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase pathway (MAPK), whereas the endogenous ligand, 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2), only activated the PI3K pathway. 15d-PGJ2 had no detectable effects on COX-2, mPGES expression, or PGE2 production. However, troglitazone induced time-dependent COX-2 expression, which was insensitive to PPAR(gamma) antagonists, but was abrogated by inhibitors of PI3K and the ERK MAP kinase pathway. Furthermore, troglitazone induced mPGES expression and PGE2 production. Neither troglitazone nor 15d-PGJ2 was able to convincingly activate NF-kappaB in A549 cells. Further heterogeneity in the responses to troglitazone and 15d-PGJ2 was observed in the regulation of gene expression as assessed by microarray analysis. In summary, this study provides compelling evidence that troglitazone (like 15d-PGJ2) can exert functional effects independently of actions via PPAR(gamma). Moreover, we have identified unique biochemical and functional actions of troglitazone that are not shared by 15d-PGJ2, which may influence the therapeutic potential of this compound in inflammatory settings.

There has been much interest recently in the antimicrobial properties of cationic peptides called beta-defensins from epithelial cells. Human beta-defensin (hBD)-1 and -2 have been particularly implicated in cystic fibrosis (CF) patients, where their inhibition by high salt concentrations may explain in part the susceptibility of the CF lung to bacterial infection. In this work, we have employed a simple co-culture system using the 16-HBE human bronchial epithelial cell line to assess growth inhibitory activity against Pseudomonas aeruginosa and Burkholderia cepacia. In medium alone, P. aeruginosa proliferated more than 100,000-fold, whereas in the presence of 16-HBE cells or 16-HBE-conditioned medium, bacterial proliferation was less than 100-fold. Raising the salt concentration of cell-free 16-HBE conditioned medium to approximately 200 mM significantly reduced this growth inhibitory activity. In contrast, there was no evidence of epithelial-derived growth inhibitory activity against two strains of B. cepacia. RT-PCR analysis indicated expression of the hBD-2 mRNA in 16-HBE cells, but not hBD-1. These data demonstrate for the first time that B. cepacia is resistant to epithelial-derived antimicrobial substances and argue against them being important in the defense against this organism in the lung.

Burkholderia (formerly Pseudomonas) cepacia has emerged as an important pulmonary pathogen in cystic fibrosis, and survives within the lung despite a vigorous neutrophil-dominated immune response. Nitric oxide (NO) contributes to the antimicrobial activity of reactive oxygen species in the normal lung, but recent evidence suggests that inducible NO synthase is not expressed in the airway epithelial cells of cystic fibrosis (CF) patients. This may explain the failure of the neutrophil response to eliminate B. cepacia. To test this hypothesis, the present study examined the combined effect of NO, superoxide and H2O2 against B. cepacia. There was no killing of a highly transmissible strain by either superoxide or NO alone, but their combination reduced the bacterial count by >1000-fold over 75 min. This bactericidal activity was not sensitive to addition of superoxide dismutase, but was abrogated completely by catalase, suggesting that NO and hydrogen peroxide were the bactericidal mediators. Increased killing by NO in combination with H2O2 was seen for seven of a further 11 strains examined. The lack of NO in the lungs of CF patients may contribute to the survival of B. cepacia.

Leukocyte accumulation and activation are key events in the pathogenesis of inflammatory lung disease. The ability of human airway smooth muscle cells (HASM) to contribute to the inflammatory process by its ability to produce the chemokines interleukin (IL) 8, monocyte chemotactic protein (MCP-1) and regulated on activation, normal T cell expressed and secreted (RANTES) was investigated. Cultured HASM, when stimulated with the pro-inflammatory cytokines IL-1 alpha (0.01-1 ng/ml) or tumour necrosis factor alpha (TNF-alpha, 0.3-30 ng/ml), synthesize and release substantial amounts of IL-8, as assessed by specific immunoassay, bioasssay (elevation of intracellular free calcium in human neutrophils), and upregulation of mRNA. These stimuli also increased MCP-1 production and mRNA expression, but RANTES mRNA expression was not detected at 24 h. The smooth muscle spasmogen endothelin 1 (1 microM) was unable to stimulate IL-8 or MCP-1 release or mRNA expression. These data indicate that HASM may constitute an important source of leukocyte attractants in the inflamed lung, where the inducing stimuli, IL-1 alpha and TNF-alpha, are also likely to be present.

1. Rheumatoid arthritis is associated with the accumulation and activation of selected populations of inflammatory cells within the arthritic joint. One putative signal for this process is the production, by resident cells, of a group of inflammatory mediators known as the chemokines. 2. The chemokines interleukin-8 (IL-8), monocyte chemotactic protein-1 (MCP-1) and RANTES (regulated on activation normal T-cell expressed and presumably secreted) are target-cell specific chemoattractants produced by synovial fibroblasts in response to stimulation with interleukin-1 alpha (IL-1 alpha) or tumour necrosis factor alpha (TNF alpha). The signalling pathways involved in their production are not well defined. We therefore used four different protein kinase C inhibitors to investigate the role of this kinase in the regulation of chemokine mRNA and protein expression in human cultured synovial fibroblasts. 3. The non-selective PKC inhibitor, staurosporine (1-300 nM) significantly increased the production of IL-1 alpha-induced IL-8 mRNA and protein. A specific PKC inhibitor, chelerythrine chloride (0.1-3 microM), also caused a small concentration-dependent increase in IL-8 mRNA and protein production. In contrast, 3-[1-[3-(amidinothio)propyl]-3-indoly]-4-(1-methyl-3-indolyl )- 1H-pyrrole-2,5-dione methanesulphonate (Ro 31-8220) and 2[1-(3-dimethylaminopropyl)-1H-indol-3-yl]-3-(1H-indol-3- yl)-maleimide (GF 109203X), two selective PKC inhibitors of the substituted bisindolylmaleimide family had a concentration-dependent biphasic effect on IL-1 alpha or TNF alpha-induced chemokine expression. At low concentrations they caused a stimulation in chemokine production, which was especially evident at the mRNA level. At higher concentrations both inhibited IL-1 alpha or TNF alpha-induced chemokine mRNA and protein production. Ro 31-8220 was 10 fold more potent than GF 109203X, with an IC50 of 1.6 +/- 0.08 microM (mean +/- s.e.mean, n = 4) for IL-1 alpha induced IL-8 production. Ro 31-8220 also inhibited the expression of IL-1 alpha or TNF alpha-induced MCP-1 and RANTES mRNA with a similar potency. 4. The stimulatory effect of staurosporine is discussed in relation to the known poor selectivity of this inhibitor for PKC. It is proposed that activation of an isoform of PKC, possibly PKC epsilon or zeta, which is inhibited by higher concentrations of the bisinodolylmaleimides, plays a role in the regulation of chemokine expression induced by IL-1 alpha or TNF alpha in synovial cells. 5. The inhibition of chemokine production by bisindolylmaleimide compounds heralds a novel approach for future anti-inflammatory therapies.

The involvement of histamine, leukotriene D4 (LTD4) and platelet-activating factor (PAF) in cutaneous anaphylaxis was investigated in a guinea pig model. When given alone, the H1 receptor antagonist chlorpheniramine, the LTD4/E4 antagonist LY171883 and the PAF antagonist WEB2086 were unable to inhibit increased microvascular plasma protein leakage in passive cutaneous anaphylaxis (PCA) reactions, as monitored by the extravasation of intravenously injected 125I-albumin. Furthermore the H2 receptor antagonist cimetidine and the serotonin antagonist methysergide were unable to reduce PCA responses when given alone or in combination with chlorpheniramine. In marked contrast, combinations of antagonists were able to reduce plasma leakage significantly. A combination of chlorpheniramine, LY171883 and WEB2086 virtually abolished plasma leakage during the PCA response, but did not influence the plasma protein leakage induced by intradermal injection of bradykinin. These results demonstrate that these allergic reactions involve several mediators and that the inability of an individual mediator antagonist to reduce responses does not necessarily rule out a role for that mediator.

Treatment of human synovial cell cultures with human recombinant interleukin 1 (IL-1) results in the appearance of activity in the supernatant which stimulates human polymorphonuclear neutrophils (PMNs), as assessed by increased chemokinesis and elevation of intracellular free calcium concentration. IL-1 (or related cytokines) were unable to stimulate these responses. This activity chromatographed as a single peak on C8 reversed-phase HPLC and subsequent size exclusion HPLC revealed two peaks of chemokinetic activity with apparent molecular masses of approximately equal to 13kDa and 6kDa. Fractions containing the higher molecular mass material also elevated PMN cytosolic free calcium. The local production of such factors may mediate IL-1-induced PMN accumulation in vivo.

Following exposure of cultured human synovial cells to human recombinant interleukin 1 alpha (IL-1 alpha), we demonstrate the appearance of factors in the supernatant which stimulate human polymorphonuclear leukocyte (PMN) locomotion and elevate intracellular free calcium ([Ca++]i). The production of these factors can be abolished by actinomycin D or dexamethasone but not by cyclo-oxygenase or lipoxygenase inhibitors. In vivo, the supernatant induces a rapid accumulation of PMNs in rabbit skin following intradermal injection. These activities were not due to IL-1 itself, tumour necrosis factor (TNF alpha) or granulocyte-macrophage colony-stimulating factor (GM-CSF). Such factors may play an important role in inflammatory responses involving IL-1.

The frequency of isolation of Burkholderia cepacia from the sputum of cystic fibrosis (CF) patients is increasing. Using the human A549 lung epithelial cell line, we have investigated the ability of B. cepacia exoproducts to stimulate interleukin-8 (IL-8) release. Cell-free supernatants from a panel of CF clinical, non-CF clinical, and nonclinical B. cepacia isolates were found to stimulate IL-8 release, with levels ranging from 11.8 +/- 2.8 to 80.0 +/- 3.5 ng/ml. A similar pattern was seen at the level of the IL-8 mRNA. The bioactivity of the IL-8 was confirmed by examining its effect on the intracellular free calcium in neutrophils and inhibition by a neutralizing anti-IL-8 antibody. B. cepacia lipopolysaccharide, which was able to stimulate IL-8 release from monocytes, did not release IL-8 from the A549 cells. Furthermore, the stimulating ability of the bacterial cell-free supernatant was not diminished by polymyxin B, was markedly reduced by boiling, and appeared unrelated to N-acylhomoserine lactones. The ability of B. cepacia to elicit IL-8 release from epithelial cells may be important in the pathology of CF.