Malcolm L. Watson

Chemokine production by human vascular smooth muscle cells: modulation by IL-13

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‐1α or tumour necrosis factor α (TNFα). 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‐1α or TNFα revealed that each stimulus induced a similar amount of MCP‐1. In contrast approximately three fold more IL‐8 was induced by IL‐1α than by TNFα whereas significant RANTES production was induced only by TNFα. These findings point to a divergence in the regulation of synthesis of the different chemokines in response to IL‐1α or TNFα 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‐1α or TNFα. 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.

Phosphoinositide 3‐kinase signalling in lung disease: leucocytes and beyond

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.

Burkholderia cepacia is resistant to the antimicrobial activity of airway epithelial cells.

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.

Arginase inhibition alleviates hypertension in the metabolic syndrome

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.

Expression and agonist responsiveness of CXCR3 variants in human T lymphocytes

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 Gαi 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.

Comparison of infrared thermography and laser speckle contrast imaging for the dynamic assessment of digital microvascular function

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 13s 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 (r(s) for pooled data between 0.5 and 0.65, p<0.00005). Reproducibility of both IRT and LSCI was excellent (ICCs>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.

Interleukin 13 Increases Contractility of Murine Tracheal Smooth Muscle by a Phosphoinositide 3-kinase p110δ-Dependent Mechanism

The Th2 cytokine interleukin (IL) 13 can elicit a number of responses consistent with a key role in the pathogenesis of asthma. We have used pharmacological and genetic approaches to demonstrate the role of signaling via the class I phosphoinositide 3-kinase p110δ isoform in IL-13-induced hyper-responsiveness of murine tracheal smooth muscle contractility in vitro. IL-13 treatment of tracheal tissue is associated with an early activation of phosphoinositide 3-kinase (PI3K), as assessed by phosphorylation of Akt. Tracheal smooth muscle contractility is enhanced by overnight incubation with IL-13, resulting in increased maximal contractions (Emax) to carbachol (CCh) and KCl. Inhibition of PI3K by the non-isoform-selective inhibitors wortmannin or 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002), or the selective inhibitor of the PI3K p110δ isoform 2-(6-aminopurin-9-ylmethyl)-5-methyl-3-O-tolyl-3H-quinazolin-4-one (IC87114), prevented IL-13-induced hyper-responsiveness. Consistent with a role for PI3K p110δ in IL-13-induced hyper-responsiveness, IL-13 was unable to induce hyper-responsiveness in tissues from mice expressing the catalytically inactive form of p110δ (p110δD910A). These data indicate that IL-13 contributes to tracheal smooth muscle hyper-responsiveness via the PI3K p110δ isoform. In addition to previously reported effects on airway inflammation, inhibition of PI3K p110δ may be a useful target for the treatment of asthma by preventing IL-13-induced airway smooth muscle hyper-responsiveness.

Differential effects of protein kinase C inhibitors on chemokine production in human synovial fibroblasts

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‐la (IL‐1α) or tumour necrosis factor α (TNFα). 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‐1α‐induced IL‐8 mRNA and protein. A specific PKC inhibitor, chelerythrine chloride (0.1–3 μm), 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 methanesul‐phonate (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‐1α or TNFα‐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‐1α or TNFα‐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 μm (mean±s.e.mean, n = 4) for IL‐1α induced IL‐8 production. Ro 31–8220 also inhibited the expression of IL‐1α or TNFα‐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‐1α or TNFα in synovial cells. 5 The inhibition of chemokine production by bisindolylmaleimide compounds heralds a novel approach for future anti‐inflammatory therapies.

Nitric oxide-induced potentiation of the killing of Burkholderia cepacia by reactive oxygen species: implications for cystic fibrosis

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.

Desensitization of the soluble guanylyl cyclase/cGMP pathway by lipopolysaccharide in rat isolated pulmonary artery but not aorta

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.