Multiple molecular recognition properties of the lipocalin protein family

The lipocalins, a diverse family of small extracellular ligand proteins, display a remarkable range of different molecular properties. While their binding of small hydrophobic molecules, and to a lesser extent their binding to cell surface receptors, is well known, it is shown here that formation of macromolecular complexes is also a common feature of this family. Analysis of known crystallographic structures reveals that the lipocalins process a conserved common structure: an antiparallel β-barrel with a repeated +1 topology. Comparisons show that within this overall similarity the structure of individual proteins is specifically adapted to bind their particular ligands, forming a binding site from an internal cavity (within the barrel) and/or an external loop scaffold, which gives rise to different binding modes that reflects the need to accommodate ligands of different shape, size, and chemical structure. The architecture of the lipocalin fold suggests that the both the ends and sides of this barrel are topologically distinct, differences also apparent in analyses of structural and sequence variation within the family. These different can be linked to experimental evidence suggesting a possible functional dichotomy between the two ends of the lipocalin fold. The structurally invariant end of the molecule may be implicated in general binding small ligands and forming macromolecular complexes via an exposed binding surface.     

Effect of canine surfactant protein (SP-A) on the respiratory burst of phagocytic cells

Cells obtained from bronchoalveolar lavage, or neutrophils of peripheral blood of dog, were incubated with the canine surfactant-associated protein A (SP-A). A significant decrease of the production of Superoxide anion was observed after subsequent stimulation with phorbol-12-myristate-13-acetate (PMA) as measured by the lucigenin-dependent chemiluminesence (CL). Several other proteins used for control experiments did not decrease lucigenin-dependent CL, indicating a specific effect of SP-A on phagocytes. Treatment of SP-A with collagenase prior to incubation with neutrophils destroyed the depleting effect on oxygen radical production of PMA-stimulated cells. We propose that SP-A acts as a regulatory factor of the respitatory burst of alveolar macrophages and neutrophils in the lungs. The inhibitory effect of SP-A is down-regulated by collagenase released from stimulated alveolar macrophages.     

Acute Phase Immune Response to Exercise Coexists with Decreased Neutrophil Antioxidant Enzyme Defences

Long-duration or damaging exercise initiates reactions that resemble the acute phase response to infection and induces neutrophil priming for oxidative activity. Our objective was to establish the status of the antioxidant defences and of the oxidative equilibrium in the neutrophils of sportsmen prior to and after intense physical exercise. Nine voluntary male professional cyclists participated in this study. The exercise was a cycling mountain stage (171 km) and the cyclists took a mean &#45 SEM of 270 &#45 12 min to complete it. We determined the activities of catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPx), the levels and activity of superoxide dismutase (SOD), the concentrations of ascorbate, glutathione and glutathione disulphide (GSSG) and DNA levels in neutrophils. The cycling stage decreased enzyme activities expressed per DNA units: CAT (33%), SOD (38%), GPx (65%); increased ascorbate concentration in neutrophils and decreased the GSH/GSSG ratio and the enzyme activities expressed per DNA units. Neutrophils could contribute to plasma antioxidant defences against oxidative stress induced by exercise because they probably provide antioxidant enzymes and ascorbate.     

Myeloperoxidase has Directly-opposed Effects on Nitration Reaction--Study on Myeloperoxidase-deficient Patient and Myeloperoxidase-knockout Mice

Myeloperoxidase (MPO) catalyzes a nitration reaction to form nitrotyrosine in the presence of high nitrite, the metabolite of NO. Human leukocyte was shown to cause phenolic nitration using released MPO as a catalyst in the presence of nitrite. It opposes our previous finding that inhibition of MPO was essential for phenol nitration in human leukocyte study. To clarify the role of MPO, we utilized MPO-deficient human leukocytes and MPO-knockout mice. Even in the absence of exogenously added nitrite, high nitration product was observed in MPO-deficient leukocytes. In liver subjected to ischemia/reperfusion injury, a significantly higher amount of nitrotyrosine was produced in MPO-knockout mice than in normal mice. These results clearly demonstrate that MPO inhibits the accumulation of nitration products in vivo . Further experiments showed that MPO could degrade nitrotyrosine in the presence of glutathione. Thus, MPO-induced degradation of nitration products may cause the underestimation of the nitration product generated in vivo . We conclude that MPO may act predominantly to scavenge nitrotyrosine under physiological nitrite condition, and protect against injurious effect of nitrotyrosine.     

Inhibition of LPS-induced airway neutrophilic inflammation in healthy volunteers with an oral CXCR2 antagonist

Background

Inhaled lipopolysaccharide (LPS) induces a dose-dependent, acute neutrophilic response in the airways of healthy volunteers that can be quantified in induced sputum. Chemokines, such as CXCL1 and CXCL8, play an important role in neutrophilic inflammation in the lung through the activation of CXCR2 and small molecule antagonists of these receptors have now been developed. We investigated the effect of AZD8309, a CXCR2 antagonist, compared with placebo on LPS-induced inflammation measured in sputum of healthy volunteers.

Methods

Twenty healthy subjects were randomized in a double-blind placebo-controlled, cross-over study. AZD8309 (300 mg) or placebo was dosed twice daily orally for 3 days prior to challenge with inhaled LPS and induced sputum was collected 6 h later.

Results

Treatment with AZD8309 showed a mean 77% reduction in total sputum cells (p < 0.001) and 79% reduction in sputum neutrophils (p < 0.05) compared with placebo after LPS challenge. There was also a reduction in neutrophil elastase activity (p < 0.05) and CXCL1 (p < 0.05) and trends for reductions in sputum macrophages (47%), leukotriene B₄ (39%) and CXCL8 (52%).

Conclusions

AZD8309 inhibited LPS-induced inflammation measured in induced sputum of normal volunteers, indicating that this treatment may be useful in the treatment of neutrophilic diseases of the airways, such as COPD, severe asthma and cystic fibrosis.

Trial registration

{"type":"clinical-trial","attrs":{"text":"NCT00860821","term_id":"NCT00860821"}}NCT00860821.     

Anti-inflammatory effects of clarithromycin in ventilator-induced lung injury

Background

Mechanical ventilation can promote lung injury by triggering a pro-inflammatory response. Macrolides may exert some immunomodulatory effects and have shown significant benefits over other antibiotics in ventilated patients. We hypothesized that macrolides could decrease ventilator-induced lung injury.

Methods

Adult mice were treated with vehicle, clarithromycin or levofloxacin, and randomized to receive mechanical ventilation with low (12 cmH₂O, PEEP 2 cmH₂O) or high (20 cmH₂O, ZEEP) inspiratory pressures for 150 minutes. Histological lung injury, neutrophil infiltration, inflammatory mediators (NFκB activation, Cxcl2, IL-10) and levels of adhesion molecules (E-selectin, ICAM) and proteases (MMP-9 and MMP-2) were analyzed.

Results

There were no differences among groups after low-pressure ventilation. Clarithromycin significantly decreased lung injury score and neutrophil count, compared to vehicle or levofloxacin, after high-pressure ventilation. Cxcl2 expression and MMP-2 and MMP-9 levels increased and IL-10 decreased after injurious ventilation, with no significant differences among treatment groups. Both clarithromycin and levofloxacin dampened the increase in NFκB activation observed in non-treated animals submitted to injurious ventilation. E-selectin levels increased after high pressure ventilation in vehicle- and levofloxacin-treated mice, but not in those receiving clarithromycin.

Conclusions

Clarithromycin ameliorates ventilator-induced lung injury and decreases neutrophil recruitment into the alveolar spaces. This could explain the advantages of macrolides in patients with acute lung injury and mechanical ventilation.     

Molecular Basis of Glycosaminoglycan Heparin Binding to the Chemokine CXCL1 Dimer

Glycosaminoglycan (GAG)-bound and soluble chemokine gradients in the vasculature and extracellular matrix mediate neutrophil recruitment to the site of microbial infection and sterile injury in the host tissue. However, the molecular principles by which chemokine-GAG interactions orchestrate these gradients are poorly understood. This, in part, can be directly attributed to the complex interrelationship between the chemokine monomer-dimer equilibrium and binding geometry and affinities that are also intimately linked to GAG length. To address some of this missing knowledge, we have characterized the structural basis of heparin binding to the murine CXCL1 dimer. CXCL1 is a neutrophil-activating chemokine and exists as both monomers and dimers (K_d = 36 μm). To avoid interference from monomer-GAG interactions, we designed a trapped dimer (dCXCL1) by introducing a disulfide bridge across the dimer interface. We characterized the binding of GAG heparin octasaccharide to dCXCL1 using solution NMR spectroscopy. Our studies show that octasaccharide binds orthogonally to the interhelical axis and spans the dimer interface and that heparin binding enhances the structural integrity of the C-terminal helical residues and stability of the dimer. We generated a quadruple mutant (H20A/K22A/K62A/K66A) on the basis of the binding data and observed that this mutant failed to bind heparin octasaccharide, validating our structural model. We propose that the stability enhancement of dimers upon GAG binding regulates in vivo neutrophil trafficking by increasing the lifetime of “active” chemokines, and that this structural knowledge could be exploited for designing inhibitors that disrupt chemokine-GAG interactions and neutrophil homing to the target tissue.     

Matrix Metalloproteinase 8 (Collagenase 2) Induces the Expression of Interleukins 6 and 8 in Breast Cancer Cells

Matrix metalloproteinase 8 (MMP-8) is a tumor-suppressive protease that cleaves numerous substrates, including matrix proteins and chemokines. In particular, MMP-8 proteolytically activates IL-8 and, thereby, regulates neutrophil chemotaxis in vivo. We explored the effects of expression of either a WT or catalytically inactive (E198A) mutant version of MMP-8 in human breast cancer cell lines. Analysis of serum-free conditioned media from three breast cancer cell lines (MCF-7, SK-BR-3, and MDA-MB-231) expressing WT MMP-8 revealed elevated levels of IL-6 and IL-8. This increase was mirrored at the mRNA level and was dependent on MMP-8 catalytic activity. However, sustained expression of WT MMP-8 by breast cancer cells was non-permissive for long-term growth, as shown by reduced colony formation compared with cells expressing either control vector or E198A mutant MMP-8. In long-term culture of transfected MDA-MB-231 cells, expression of WT but not E198A mutant MMP-8 was lost, with IL-6 and IL-8 levels returning to base line. Rare clonal isolates of MDA-MB-231 cells expressing WT MMP-8 were generated, and these showed constitutively high levels of IL-6 and IL-8, although production of the interleukins was no longer dependent upon MMP-8 activity. These studies support a causal connection between MMP-8 activity and the IL-6/IL-8 network, with an acute response to MMP-8 involving induction of the proinflammatory mediators, which may in part serve to compensate for the deleterious effects of MMP-8 on breast cancer cell growth. This axis may be relevant to the recognized ability of MMP-8 to orchestrate the innate immune system in inflammation in vivo.