Effects of ion composition and tonicity on human neutrophil antibacterial activity

Infants with cystic fibrosis (CF) often are infected with Staphylococcus aureus (S. aur.), which is followed by colonization with Pseudomonas aeruginosa (P. aerug.). In spite of an excessive, neutrophil-dominated inflammatory response in the respiratory tract, patients with CF often succumb to pulmonary infections with P. aerug. Because peripheral blood neutrophils of these patients have normal functions, we examined whether hypothesized alterations of the airway surface liquids (ASL) in these patients significantly impair neutrophil bactericidal activity in the microenvironment of the CF lung. The ionic composition of CF ASL is still not entirely defined and has been speculated to be abnormally high or abnormally low in Na+ and Cl- concentrations; estimates of osmolarities have ranged from 200 (hypo-osmolar) to 285 (iso-osmolar) to > 300 meq/L (hyper-osmolar). Our data indicate that bacterial killing activity of human peripheral blood neutrophils against P. aerug. or S. aur. is not decreased in buffers in which NaCI was replaced with equimolar concentrations of choline Cl. KC1, or N-methyl-D-glucamine chloride to maintain isotonicity. Amiloride or benzamil, known modulators of Na+ transport in neutrophils, did not interfere with this neutrophil function. Deviations from isotonicity of ± 50% also failed to diminish bactericidal activity of neutrophils significantly. In contrast, superoxide production and enzyme secretion in response to the chemotactic peptide N-formylmethionylleucylphenylalanine appeared to be sensitive to the ionic milieu of the assay buffers. Our results suggest that the postulated alterations in the ionic composition of ASL in CF lungs are insufficient to explain why neutrophils fail to clear infections with P. aerug. in these patients

CD14-dependent lipopolysaccharide-induced β-defensin-2 expression in human tracheobronchial epithelium

The induction of host antimicrobial molecules following binding of pathogen components to pattern recognition receptors such as CD14 and the Toll-like receptors (TLRs) is a key feature of innate immunity. The human airway epithelium is an important environmental interface, but LPS recognition pathways have not been determined. We hypothesized that LPS would trigger β-defensin (hBD2) mRNA in human tracheobronchial epithelial (hTBE) cells through a CD14-dependent mechanism, ultimately activating NF-κB. An average 3-fold increase in hBD2 mRNA occurs 24 h after LPS challenge of hTBE cells. For the first time, we demonstrate the presence of CD14 mRNA and cell surface protein in hTBE cells and show that CD14 neutralization abolishes LPS induction of hBD2 mRNA. Furthermore, we demonstrate TLR mRNA in hTBE cells and NF-κB activation following LPS. Thus, LPS induction of hBD2 in hTBE cells requires CD14, which may complex with a TLR to ultimately activate NF-κB

Function and regulation of chemoattractant receptors

Phagocyte migration and activation at sites of inflammation is mediated through chemoattractant receptors that are coupled to G-proteins. Early studies from our laboratory demonstrated G-protein-mediated phospholipase C activation by chemoattractants. Recently, this laboratory developed cellular and animal models to allow biochemical, cell biological and molecular genetic approaches to be used in determining the mechanisms of chemoattractant receptor function, regulation, and cross regulation. These studies provided evidence that chemoattractant receptors activate distinct pathways for chemotaxis and exocytosis and cross-regulate each other's function at multiple levels. A major site of regulation is through phosphorylation of receptors by G-protein-coupled receptor kinases and by protein kinase C. In addition, the activation of phospholipase C by chemoattractants is also regulated, at additional sites distal to receptor phosphorylation. These may include modulation of G-protein activation by regulators of G-protein signaling (RGS) and modification of phospholipase C. Phosphorylation of phospholipase Cβ3 by both protein kinase A and protein kinase C has been demonstrated. The function and regulation of chemoattractant receptors are also being examined in mouse models. In these studies, mice deficient in leukotriene B4 receptors have been generated by targeted gene disruption. These mice displayed reduced neutrophil accumulation in certain inflammation models and sex-related differences in platelet-activating-factor induced anaphylaxis

An unrecognized ancient lineage of green plants persists in deep marine waters

We provide molecular phylogenetic evidence that the obscure genera Palmophyllum Kütz. and Verdigellas D. L. Ballant. et J. N. Norris form a distinct and early diverging lineage of green algae. These palmelloid seaweeds generally persist in deep waters, where grazing pressure and competition for space are reduced. Their distinctness warrants recognition as a new order, the Palmophyllales. Although phylogenetic analyses of both the 18S rRNA gene and two chloroplast genes (atpB and rbcL) are in agreement with a deep-branching Palmophyllales, the genes are in conflict about its exact phylogenetic placement. Analysis of the nuclear ribosomal DNA allies the Palmophyllales with the prasinophyte genera Prasinococcus and Prasinoderma (Prasinococcales), while the plastid gene phylogeny placed Palmophyllum and Verdigellas as sister clade to all other Chlorophyta

A Selective Phosphodiesterase-4 Inhibitor Reduces Leukocyte Infiltration, Oxidative Processes, and Tissue Damage after Spinal Cord Injury

We tested the hypothesis that a selective phosphodiesterase type 4 inhibitor (PDE4-I; IC486051) would attenuate early inflammatory and oxidative processes following spinal cord injury (SCI) when delivered during the first 3 days after injury. Rats receiving a moderately severe thoracic-clip-compression SCI were treated with the PDE4-I (0.5, 1.0, and 3.0 mg/kg IV) in bolus doses from 2–60 h post-injury. Doses at 0.5 mg/kg and 1.0 mg/kg significantly decreased myeloperoxidase (MPO) enzymatic activity (neutrophils), expression of a neutrophil-associated protein and of ED-1 (macrophages), and estimates of lipid peroxidation in cord lesion homogenates at 24 h and 72 h post-injury by 25–40%. The 3.0 mg/kg dose had small or no effects on these measures. The PDE4-I treatment (0.5 or 1.0 mg/kg) reduced expression of the oxidative enzymes gp91phox, inducible nitric oxide synthase, and cyclooxygenase-2, and diminished free radical generation by up to 40%. Treatment with 0.5 mg/kg PDE4-I improved motor function (as assessed by the Basso-Beattie-Bresnahan scale) significantly from 4–8 weeks after SCI (average difference 1.3 points). Mechanical allodynia elicited from the hindpaw decreased by up to 25%. The PDE4-I treatment also increased white matter volume near the lesion at 8 weeks after SCI. In conclusion, the PDE4-I reduced key markers of oxidative stress and leukocyte infiltration, producing cellular protection, locomotor improvements, and a reduction in neuropathic pain. Early inhibition of PDE4 is neuroprotective after SCI when given acutely and briefly at sufficient doses