Neutrophil collagenase in sputum from patients with cystic fibrosis

The potential role of neutrophil elastase in causing lung damage and exacerbating the inflammatory response in cystic fibrosis (CF) has received considerable attention. Although another potent neutrophil-derived enzyme, collagenase, is implicated in tissue destruction in several interstitial lung disorders, there has been no reference to this enzyme in CF. The objective of this study was to determine whether neutrophil collagenase is present in active form in CF sputum and, if so, whether it is related to disease severity. High levels of active collagenase were detected in sputum from patients with CF, and the majority of the enzyme present was of neutrophil origin. In a group of 16 patients with CF, negative relations between sputum collagenase activity and Shwachman score (r = -0.55, p < 0.05) and FEV1 (r = -0.59, p < 0.02) were noted, indicating an association between high collagenase activity and severity of disease. A positive correlation was observed between sputum collagenase and elastase activity (r = 0.62, p < 0.05). These results suggest that both neutrophil elastase and collagenase may play a significant role in lung destruction in CF.     

Activation of neutrophil respiratory burst by cytokines and chemoattractants. Regulatory role of extracellular matrix glycoproteins

OBJECTIVE AND DESIGN: We investigated the in vitro responsiveness of neutrophils adherent to fibronectin (FN) and laminin (LM), toward natural pro-inflammatory and/or phagocyte-activating agents. MATERIALS AND METHODS: Neutrophils from normal volunteers were layered on polystyrene wells precoated or not with FN and/or LM and tested for their ability of responding to eleven pro-inflammatory mediators by evaluation of superoxide anion (O2-) production and adherence. Results, expressed as mean +/-1SEM, were evaluated by non-parametric analyses (Mann-Whitney U-test or Kruskal-Wallis non-parametric ANOVA analysis) RESULTS: Precoating polystyrene wells with LM or FN prevented the plastic-induced neutrophil (O2-) production. Among eleven agents, tumor necrosis factor-alpha (TNF, 3.0+/-0.3 nmoles (O2-)/5 x 10(4) neutrophils/180 min, p < 0.001), granulocyte-macrophage colony stimulating factor (GM-CSF, 2.1+/-0.3 nmoles (O2-)/5 x 10(4) neutrophils/180 min, p < 0.05) and formyl-peptides (fMLP, 2.5+/-0.5 nmoles (O2-)/5 x 10(4) neutrophils/180min, p < 0.01) caused massive (O2-) production by neutrophils adherent to FN. None of the mediators was capable of triggering (O2-) production by neutrophils adherent to LM. LM, mixed with FN to coat wells, caused a dose-dependent inhibition of the oxidative burst triggered by TNF (IC50 LM: 0.84+/-0.03 microg, mean+/-1 SEM), GM-CSF (IC50 LM: 0.36+/-0.16micro/g, mean+/-1SEM) and fMLP (IC50 LM: 0.54+/-0.008 microg, mean+/-1 SEM). To the contrary, fMLP (85.5+/-27.7%), TNF (163.1+/-67.5%), and GM-CSF (121.8+/-66.4%) caused a significant augmentation of neutrophil adherence to LM, suggesting that LM-mediated inhibition of neutrophil oxidative metabolism does not depend on the concomitant LM-induced inhibition of neutrophil adherence. Finally, neither solid-phase FN nor LM affected (O2-) production by neutrophils in response to immune complexes. CONCLUSIONS: Extracellular matrix glycoproteins dictate the response of neutrophils to soluble mediators but not to immune complexes. This appears to be a biologically meaningful mechanism to localise the risk of cellular reactions to mediators that are able to diffuse easily from tissue sites of generation and become widely distributed in body fluids during inflammatory diseases.     

The Yin and Yang of oxidation in the development of the fatty streak. A review based on the 1994 George Lyman Duff Memorial Lecture

Recent data support the hypothesis that the fatty streak develops in response to specific phospholipids contained in LDL that become trapped in the artery wall and become oxidized as a result of exposure to the oxidative waste of the artery wall cells. The antioxidants present within both LDL and the microenvironments in which LDL is trapped function to prevent the formation of these biologically active, oxidized lipids. Enzymes associated with LDL and HDL (eg, platelet activating factor acetylhydrolase) or with HDL alone (eg, paraoxonase) destroy these biologically active lipids. The regulation and expression of these enzymes are determined genetically and are also significantly modified by environmental influences, including the acute-phase response or an atherogenic diet. The balance of these multiple factors leads to an induction or suppression of the inflammatory response in the artery wall and determines the clinical course.     

Effect of serum amyloid A on selected in vitro functions of isolated human neutrophils

Serum amyloid A (SAA) is an acute phase reactant whose levels in the blood rise as part of the body's response to stress and inflammation. Previous studies have suggested that SAA may carry an anti-inflammatory potential. We evaluated the effects of SAA on human neutrophils activated by N-formyl-methionyl-leucyl-phenylalanine (fMLP) in vitro. At concentrations higher than 10 microg/mL, SAA inhibited neutrophil myeloperoxidase (MPO) release. This effect was located in the N-terminal--that is, amino acid residues 1-14--of the SAA molecule. Directed neutrophil migration was inhibited at the same SAA concentrations. Several amino acid residues (1-14, 15-104, 83-104) contributed to this effect. Neutrophil O2- production was inhibited at low concentrations of SAA (0.1 to 1 microg/ml) and was stimulated at concentrations higher than 50 microg/mL. Neutrophil O2- production induced by phorbol myristate acetate (PMA) and O2- generated by the xanthine-xanthine oxidase reaction were not affected by SAA. These results add to previous data suggesting that SAA, at concentrations recorded in the serum during inflammation, modulates neutrophil function; thus it may play a role in the down-regulation of the inflammatory process.     

Cellular localization of the inhibitory action of abruquinone A against respiratory burst in rat neutrophils

1. The possible mechanisms of action of the inhibitory effect of abruquinone A on the respiratory burst in rat neutrophils in vitro was investigated. 2. Abruquinone A caused an irreversible and a concentration-dependent inhibition of formylmethionylleucyl-phenylalanine (fMLP) plus dihydrocytochalasin B (CB)- and phorbol 12-myristate 13-acetate (PMA)-induced superoxide anion (O2.-) generation with IC50 values of 0.33 +/- 0.05 microgram ml-1 and 0.49 +/- 0.04 microgram ml-1, respectively. 3. Abruquinone A also inhibited O2 consumption in neutrophils in response to fMLP/CB and PMA. However, abruquinone A did not scavenge the generated O2.- in xanthine-xanthine oxidase system and during dihydroxyfumaric acid (DHF) autoxidation. 4. Abruquinone A inhibited both the transient elevation of [Ca2+]i in the absence of [Ca2+]o (IC50 7.8 +/- 0.2 micrograms ml-1) and the generation of inositol trisphosphate (IP3) (IC50 10.6 +/- 2.0 micrograms ml-1) in response to fMLP. 5. Abruquinone A did not affect the enzyme activaties of neutrophil cytosolic protein kinase C (PKC) and porcine heart protein kinase A (PKA). 6. Abruquinone A had no effect on intracellular guanosine 3':5'-cyclic monophosphate (cyclic GMP) levels but decreased the adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels. 7. The cellular formation of phosphatidic acid (PA) and phosphatidylethanol (PEt) induced by fMLP/ CB was inhibited by abruquinone A with IC50 values of 2.2 +/- 0.6 micrograms ml-1 and 2.5 +/- 0.3 micrograms ml-1, respectively. Abruquinone A did not inhibit the fMLP/CB-induced protein tyrosine phosphorylation but induced additional phosphotyrosine accumulation on proteins of 73-78 kDa in activated neutrophils. 8. Abruquinone A inhibited both the O2.- generation in PMA-activated neutrophil particulate NADPH oxidase (IC50 0.6 +/- 0.1 microgram ml-1) and the iodonitrotetrazolium violet (INT) reduction in arachidonic acid (AA)-activated cell-free system (IC50 1.5 +/- 0.2 micrograms ml-1) 9. Collectively, these results indicate that the inhibition of respiratory burst in rat neutrophils by abruquinone A is mediated partly by the blockade of phospholipase C (PLC) and phospholipase D (PLD) pathways, and by suppressing the function of NADPH oxidase through the interruption of electron transport.     

Early detection and control of risk factors in diabetic nephropathy

An influx of neutrophils into the airways is a common feature observed during pulmonary inflammation induced by air pollutants, including sulfur dioxide and sulfates. In the present study focusing on the in vitro interactions of sodium sulfite (Na2SO3) with human neutrophils, we confirm results indicating that this sulfite induces superoxide production (O2-) by itself. We demonstrated that this response can occur more rapidly than previously reported (within 5 min), and that Na2SO3 can act as a priming agent, in a concentration-dependent fashion, to the bacterial tripeptide N-formyl-methionine-leucine-phenylalanine (fMLP) by increasing O2-production. In addition, our results show that Na2SO3 induces gene expression in human neutrophils in a concentration-dependent manner as assessed by incorporation of 5-[3H] uridine into total RNA. However, it does not induce cell shape changes. We also demonstrated that Na2SO3 does not modulate neutrophil apoptosis nor reverse the well-known delaying effect of granulocyte-macrophage colony-stimulating factor (GM-CSF) on apoptosis. We conclude that Na2SO3 acts rapidly on neutrophil physiology, within a few minutes with respect to superoxide production, and a few hours (4 h) with respect to gene expression without altering a biological process such as the rate of apoptosis evaluated after a long period of incubation (20 h). We further conclude that Na2SO3-induced production of O2does not drive neutrophils to undergo apoptosis, a mechanism known to occur in other conditions. Therefore, the potential toxicity of Na2SO3 during pulmonary inflammation or lung-associated diseases may be related to its ability to induce superoxide production without altering neutrophil apoptosis rate.     

Highly potent irreversible inhibitors of neutrophil elastase generated by selection from a randomized DNA-valine phosphonate library

We incorporated a phosphonate irreversible inhibitor of neutrophil elastase into a randomized DNA library and, using the SELEX process, iteratively selected these assemblies for the most potent elastase inhibitors. The inhibitors were selected against purified elastase and against secreted elastase in the presence of activated neutrophils. Very active aptamer inhibitors were obtained by both methods, with second-order rate constants for inactivation of human neutrophil elastase ranging (1-3) x 10(8) M(-1) min(-1). These rates exceed those of any reported irreversible inhibitor of elastase and exceed the previous best phosphonate inhibitors by 80-fold. The selected inhibitors are also significantly more potent than alpha-1 proteinase inhibitor in blocking degradation of elastin by activated neutrophils. In contrast to a previous experiment [Smith et al. (1995) Chem. Biol. 2, 741-750], a single-enantiomer form of the valyl phosphonate was used rather than a racemic mixture. Our analysis shows that this use of a chirally resolved valyl phosphonate results in selection of much more potent inhibitors and that these inhibitors specifically potentiate a single enantiomeric form of the phosphonate.     

Neutrophil migration, oxidative metabolism, and adhesion in elderly and young subjects

OBJECTIVE: To evaluate neutrophil functions in the elderly. METHODS: We investigated the PMN migration in vivo and PMN superoxide production and adhesion in response to a variety of compounds; PMN have been isolated both from blood and from a skin experimental exudate (obtained by Senn's skin window technique) of 25 normal elderly and of 25 normal young control subjects. RESULTS: No difference was found in PMN migration in vivo (62.9 +/- 21.3 x 10(6) and 65.5 +/- 9.1 x 10(6) PMN/cm2/24 hours in elderly and young subjects respectively), neither were different the adhesion under basal condition and after some stimuli and the superoxide production in basal condition and in response to STZ and PMA in two groups. In elderly subjects superoxide production, in response to fMLP, markedly resulted lower than in young controls both by circulating PMNs (3.6 +/- 2.7 and 9.3 +/- 3.3 nMOLES O2-/10(6) PMN respectively, p < 0.0001) and by exudate PMNs (13.6 +/- 4.3 and 19.4 +/- 6 nMOLES O2-/10(6) PMNs respectively, p < 0.005). CONCLUSION: Many PMN functions in the elderly do not differ from young people, suggesting that the overall defense function of these cells is not affected by aging. The only parameter that we have found to be different between the two groups is the poor superoxide production after fMLP stimulus of PMNs. The stimulus- and function-specificity of this defect in PMNs from elderly subjects indicates the existence of a dysregulation of the signal transduction pathway distal to fMLP receptor and proximal to NADPH oxidase activation.     

Characterization of the inhibitory prostanoid receptors on human neutrophils

1. We have evaluated the effects of various prostanoid agonists on the release of leukotriene B4 (LTB4) and superoxide anions (O2-) from human neutrophils stimulated with opsonized zymosan (OZ) and formyl-methionyl-leucyl-phenylalanine (FMLP), respectively. 2. Prostaglandin E2 (PGE2) and PGD2 inhibited both OZ-induced LTB4 release (EC50 0.72 microM and 0.91 microM respectively), and FMLP-induced O2- release (EC50 0.42 microM and 0.50 microM respectively). PGF2 alpha, the TP-receptor agonist, U46619, and the IP-receptor agonist, iloprost, were also active, but were all at least an order of magnitude less potent than PGE2 and PGD2. 3. The EP2/EP3-receptor agonist, misoprostol, and the selective EP2-agonist, AH13205, were both effective inhibitors of LTB4 release, being approximately equipotent with and 16-times less potent than PGE2, respectively. In contrast, the EP1/EP3-receptor agonist, sulprostone, had no inhibitory activity at concentrations of up to 10 microM. 4. The selective DP-receptor agonist, BW245C, inhibited LTB4 release, (EC50 0.006 microM) being approximately 50 times more potent than PGD2. BW245C also inhibited O2- release, and this inhibition was antagonized competitively by the DP-receptor blocking drug, AH6809 (pA2 6.6). 5. These data indicate the presence of both inhibitory EP- and DP-receptors on the human neutrophil. The rank order of potency of EP-receptor agonists suggest that the EP-receptors are of the EP2-subtype.     

Novel C-terminally truncated isoforms of the CXC chemokine beta-thromboglobulin and their impact on neutrophil functions

The neutrophil agonist neutrophil-activating peptide-2 (NAP-2) arises through proteolytic processing of platelet-derived N-terminally extended inactive precursors, the most abundant one being connective tissue-activating peptide-III (CTAP-III). Apart from N-terminal processing, C-terminal processing also appears to participate in the functional regulation of NAP-2, as indicated by our recent identification of an isoform missing four C-terminal amino acids, NAP-2 (1-66), which was about threefold more potent than full-size NAP-2. In the present study, we report on the discovery and identification of natural NAP-2 (1-63), an isoform truncated by seven C-terminal residues. Functional and receptor-binding analyses demonstrated that NAP-2 (1-63) represents the most active isoform, being about fivefold more potent than full-size NAP-2. Analyses of rNAP-2 isoforms successively truncated at the C terminus by up to eight residues suggest functionally important roles for acidic residues and for the leucine at position 63, a residue highly conserved within CXC chemokines. Finally, we report on a novel C-terminally truncated isoform of CTAP-III (CTAP-III (1-81)) representing the potential precursor of NAP-2 (1-66). We show that C-terminal truncation in CTAP-III enhances its potency to desensitize chemokine-induced neutrophil activation, indicating that C-terminal processing might not only serve to enhance neutrophil activation, but might as well participate in the down-regulation of an inflammatory response.     

Lipocortin 1 and chemokine modulation of granulocyte and monocyte accumulation in experimental inflammation

1. Migration of blood-derived leukocytes to tissue sites of inflammation is a hallmark of the response that the host organizes to counteract an insult or a trauma or an infection. A cascade of events is then activated to allow interaction between the leukocyte and the endothelium of postcapillary venule, and this cascade is finely regulated such that mechanisms of negative control are operating side by side with pathways that promote and sustain the extravasation process. Examples of both these positive and negative regulatory systems are discussed here. 2. In vivo accumulation of specific subtypes of leukocytes in response to application of selective chemokines operates through an indirect mechanism that includes the perivenular mast cell and, in particular, the mast cell-derived amines, such as histamine and serotonin. In fact, treatments of animals with (1) histamine H1 or serotonin antagonists or with (2) the mast cell stabilizer cromolyn or with (3) prior depletion of intact mast cells are maneuvers that successfully reduce eosinophil, neutrophil and monocyte extravasation in response to eotaxin, interleukin-8 or monocyte chemoattractant protein-1, respectively. A model in which histamine provides a P-selectin-dependent rolling phenomenon is then postulated. 3. The discovery that neutrophil-derived lipocortin 1 acts as an autocrine mediator with an inhibitory action on the emigration (diapedesis) process confirms the growing body of experimental data that showed that exogenously administered lipocortin 1 and lipocortin 1 mimetics (peptide Ac2-26) potently inhibit neutrophil extravasation in response to different stimuli. Externalization of lipocortin 1 on the plasma membrane of adherent neutrophils reduces their rate of passage through the endothelial gaps. Because cell-associated lipocortin 1 levels are under the partial control of corticosterone (endogenous circulating glucocorticoid hormone in rodents) and dexamethasone (a synthetic glucocorticoid hormone with a potent anti-inflammatory profile), a model is proposed in which a balance between anti-inflammatory (lipocortin 1, etc.) and pro-inflammatory (adhesion molecules, cytokines and chemokines) mediators explains the difference in the rate of leukocyte accumulation during the different stages of the host inflammatory response. 4. In conclusion, this review emphasizes the importance of in vivo experimental systems as a valid way of obtaining pertinent observations and reiterates the importance of negative regulatory mechanisms on the leukocyte extravasation process operating within the host.     

Identification of defensin-1, defensin-2, and CAP37/azurocidin as T-cell chemoattractant proteins released from interleukin-8-stimulated neutrophils

Reports that interleukin-8 (IL-8) induces the infiltration of neutrophils followed by T-cells into injection sites led us to postulate that by stimulation of neutrophil degranulation IL-8 may cause the release of factors with chemoattractant activity for T-lymphocytes. Extracts of human neutrophil granules were chromatographed to isolate and purify T-lymphocyte chemoattractant factors. Two major peaks of T-cell chemotactic activity were purified by C18 reversed phase high pressure liquid chromatography (HPLC). The first peak was resolved further by C4 reversed phase HPLC and yielded an active fraction shown by NH2-terminal amino acid sequence analysis to contain defensins HNP-1, HNP-2, and HNP-3. Purified defensins HNP-1 and HNP-2 (kindly provided by Dr. R. I. Lehrer, UCLA) were also potent chemoattractants for human T-cells, while HNP-3 was inactive. The second peak of T-cell chemoattractant activity was also further purified to homogeneity by C4 reversed phase HPLC and identified by NH2-terminal sequence analysis as CAP37/azurocidin, a protein with sequence homology to serine proteases. 0.1 100 ng of defensins and 1.0 100 ng/ml CAP37 were able to stimulate in vitro T-cell chemotaxis. Neutrophil activating factors, i.e. IL-8, phorbol 12-myristate 13-acetate/ionomycin, and formylmethionylleucylphenylalanine each induced the release of CAP37 and defensins from neutrophil granules. Subcutaneous administration of defensins or CAP37/azurocidin into BALB/c mice resulted in a moderate neutrophil and mononuclear cell infiltrate by 4 h, which was greater by 24 h at the site of injection. Additionally, subcutaneous injection of defensins into chimeric huPBL-SCID mice resulted in significant infiltration by human CD3+ cells within 4 h. These results identify the antimicrobial proteins, CAP37/azurocidin and defensins HNP-1 and HNP-2, as potent neutrophil-derived chemoattractants for T-cells. These proteins represent primordial antimicrobial peptides which may have evolved into acute inflammatory cell-derived signals that mobilize immunocompetent T-cells and other inflammatory cells.     

In vivo suppression of immune complex-induced alveolitis by secretory leukoproteinase inhibitor and tissue inhibitor of metalloproteinases 2

The pulmonary tree is exposed to neutrophil-derived serine proteinases and matrix metalloproteinases in inflammatory lung diseases, but the degree to which these enzymes participate in tissue injury remains undefined, as does the therapeutic utility of antiproteinase-based interventions. To address these issues, an in vivo rat model was examined in which the intrapulmonary deposition of immune complexes initiates a neutrophil-mediated acute alveolitis. In vitro studies demonstrated that rat neutrophils can release neutrophil elastase and cathepsin G as well as a neutrophil progelatinase, which was subsequently activated by either chlorinated oxidants or serine proteinases. Based on structural homologies that exist between rat and human neutrophil proteinases, rat neutrophil elastase and cathepsin G activities could be specifically regulated in vitro by recombinant human secretory leukoproteinase inhibitor, and rat neutrophil gelatinase activity proved sensitive to inhibition by recombinant human tissue inhibitor of metalloproteinases 2. When either of the recombinant antiproteinases were instilled intratracheally, in vivo lung damage as assessed by increased permeability or hemorrhage was significantly reduced. Furthermore, the coadministration of the serine and matrix metalloproteinase inhibitors almost completely prevented pulmonary damage while effecting only a modest decrease in neutrophil influx. These data support a critical role for neutrophil-derived proteinases in acute lung damage in vivo and identify recombinant human secretory leukoproteinase and recombinant human tissue inhibitor of metalloproteinases 2 as potentially efficacious interventions in inflammatory disease states.     

Changes of neutrophil migration without modification of in vitro metabolism and adhesion in Beh?et's disease

OBJECTIVE: Increase of neutrophil chemotaxis in Beh?et's disease (BD) has been described, but it is not clear whether there is a correlation with other variables of neutrophil function and whether these modifications correlate with disease activity. METHODS: We studied neutrophil functions in patients with BD in the acute phase in comparison with healthy control subjects and with the same patients during disease remission, with or without therapy. We investigated in vivo neutrophil migration by Senn's skin window technique and measured adhesion assay and superoxide production in circulating and migrating neutrophils after different stimuli. RESULTS: Neutrophil migration in vivo was 101.3 +/- 17.9 x 10(6) polymorphonuclear lymphocytes (PMN)/cm2/24 h in patients with BD in the acute phase and 66.1 +/- 7.8 x 10(6) PMN/cm2/24 h in controls (p < 0.001). No correlation was found between leukocyte counts and neutrophil migration. Neutrophil migration evaluated in the same patients in a phase of disease remission was 58.3 +/- 10.3 x 10(6) PMN/cm2/24 h (p < 0.001 vs acute phase, not significant vs controls). The neutrophils of the exudate were normally primed to response to the chemotactic peptide fMLP. No differences between the 2 groups were found in superoxide production, adhesion under basal conditions, or in response to different stimuli by circulating and migrating neutrophils. CONCLUSION: Abnormally high migration of neutrophils in the active phase of BD is the only consistent neutrophil dysfunction. Since this modification is reversed by therapy, the evaluation of in vivo neutrophil migration may be useful in diagnosing and monitoring disease activity. Blood neutrophils have normal responses to different stimuli, indicating they are not primed by the disease state.     

Regulation of guanosine 3':5'-cyclic monophosphate in ovine tracheal epithelial cells

1. Guanosine 3':5'-cyclic monophosphate (cyclic GMP) is an important second messenger mediating the effects of nitric oxide (NO) and natriuretic peptides. Cyclic GMP pathways regulate several aspects of lung pathophysiology in a number of airway cells. The regulation of this system has not been extensively studied in pulmonary epithelial tissue. 2. We have studied the production of cyclic GMP by suspensions of ovine tracheal epithelial cells in response to activators of soluble guanylyl cyclase (sodium nitroprusside (SNP) and S-nitroso-N-acetyl-penicillamine (SNAP) and particulate guanylyl cyclase (atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and E. coli heat stable enterotoxin (STa)). 3. Both 10(-7)-10(-3) M and 10(-7)-10(-3) M SNAP generated a concentration-dependent marked elevation in cyclic GMP production when incubated with 10(-3) M 3-isobutyl-l -methylxanthine (IBMX) (both greater than 25 x baseline values with highest drug concentration). 4. The increase in production of cyclic GMP in response to 10(-6) M SNP and 10(-5) M SNAP was markedly inhibited by both 5 x 10(-5) M haemoglobin (102% and 92% inhibition) and 5 x 10(-5) M methylene blue (82% and 84% inhibition). 5. The increase in cyclic GMP in response to 10(-3) M SNP was measured following co-incubation with the phosphodiesterase inhibitors 10(-7)-10(-3) M IBMX, 10(-7)-10(-4) M milrinone and 10(-7)-10(-4) M SKF 96231. Only 10(-4)-10(-3) M IBMX significantly increased cyclic GMP levels. 6. Cyclic GMP production was also significantly elevated from baseline by 10(-5) M ANP, 10(-5) M BNP, 10(-5) M CNP and 200 iu ml-3 of E. coli STa toxin in the presence of 10(-3) M IBMX. Increases with these natriuretic peptides and STa toxin were smaller in magnitude (2-4 fold) than those seen with SNP and SNAP. CNP was the most potent of the natriuretic peptides studied suggesting type B membrane bound guanylate cyclase is the predominant form expressed. 7. These results suggest that ovine tracheal epithelial cells contain active guanylyl cyclases. The more marked response to SNP and SNAP than to natriuretic peptides suggests that soluble guanylyl cyclase predominates.     

Lyme disease--on the basis of our observations

We biologically assessed functions of several reconstituted surfactants with the same minimum surface tension (2-3 mN/m) as "complete" porcine pulmonary surfactant (natural surfactant) but with longer surface adsorption times. Administration of natural surfactant (adsorption time 0.29 s) into the lungs of surfactant-deficient immature rabbits brought a tidal volume of 16.1 +/- 4.4 (SD) ml/kg during mechanical ventilation with 40 breaths/min and 20 cmH2O insufflation pressure. In static pressure-volume recordings, these animals showed a lung volume of 62.4 +/- 9.7 ml/kg at 30 cmH2O airway pressure and maintained 55% of this volume when the pressure decreased to 5 cmH2O. With two reconstituted surfactants consisting of synthetic lipids or isolated lipids from porcine lungs plus surfactant-associated hydrophobic proteins (adsorption times 0.57 and 0.78 s, respectively), tidal volumes were < 38% of that with natural surfactant (P < 0.05), but static pressure-volume recordings were not different. Care is therefore needed in estimating the in vivo function of surfactant preparations from minimum surface tension or static pressure-volume measurements.     

TNF-induced superoxide anion production in adherent human neutrophils involves both the p55 and p75 TNF receptor

TNF, a potent activator of neutrophil granulocytes, acts via two cell-surface receptors: the p55-TNF receptor (TNF-R55) and the p75-TNF receptor (TNF-R75), which can be cleaved from the cell surface and thus form soluble TNF-binding proteins (TNF-BP). The role of the two receptors in activation of the neutrophil respiratory burst was investigated. Two mAbs reacting with TNF-R55 (H398 and TBP2) induced O2 release in a similar manner but to a lesser extent than TNF. TBP2, however, required preincubation at 4 degrees C to exert its effect. Preincubation of neutrophils (both at 4 and 37 degrees C) with mAb to TNF-R75 decreased TNF-induced superoxide anion production by 67 and 64%, respectively, indicating the essential role also for TNF-R75 in neutrophil activation. This inhibitory effect could not be explained by cross-down-regulation of TNF-R55 because the TNF-R75 mAb had no effect on TNF binding to TNF-R55 as determined by binding of 125I-labeled TNF or release of TNF-R55-BP as measured by ELISA. Furthermore, the TNF-R75 mAb did not decrease superoxide anion generation induced by the TNF-R55 mAb H398, thus ruling out that the inhibitory effect of the TNF-R75 mAb is due to inhibition of the signaling pathway downstream of TNF-R55. In contrast to the TNF-R75 mAb, TNF-R55 mAbs induced down-regulation of TNF-R75 and shedding of both TNF-R55-BP and TNF-R75-BP. We conclude that both TNF-R55 and TNF-R75 are involved in TNF-induced activation of the neutrophil respiratory burst.     

Formation of a novel 20-hydroxylated metabolite of lipoxin A4 by human neutrophil microsomes

Lipoxin A4 (LXA4) is a biologically active compound produced from arachidonic acid via interactions of lipoxygenases. Incubation of LXA4 either with human neutrophils or with the neutrophil microsomes leads to formation of a polar compound on a reverse-phase high-performance liquid chromatography. We have identified the metabolite as 20-hydroxy-LXA4, a novel metabolite of arachidonic acid, on the basis of ultraviolet spectrometry and gas chromatography-mass spectrometry. The LXA4 omega-hydroxylation requires both molecular oxygen and NADPH, and is inhibited by carbon monoxide, by antibodies raised against NADPH-cytochrome P-450 reductase, or competitively by leukotriene B4 (LTB4) and LTB5, substrates of LTB4 omega-hydroxylase. These findings indicate that the formation of 20-hydroxy-LXA4 is catalyzed by a neutrophil cytochrome P-450, the LTB4 omega-hydroxylase.     

Comparative mapping of Xp22 genes in hominoids--evolutionary linear instability of their Y homologues

The extracellular matrix is now recognized as a biologically active and dynamic composition of structural, adhesive, and counteradhesive fibrous proteins embedded in a hydrated ground substance of glycosaminoglycans and proteoglycans. The ability of resident cells to detect small differences in the specific combination, concentration and distribution of matrix components suggests that perturbation of the homeostatic matrix can lead to remodelling following angioplasty. Recent studies reviewed herein have focused on how alterations of the relative composition of matrix components ultimately leads to changes in cell growth, behaviour and differentiation, all of which can significantly contribute to remodelling of the vascular wall following injury. These cell-matrix interactions may provide novel therapeutic targets in the prevention of unfavourable remodelling that leads to restenosis.