Autophagy of mucin granules contributes to resolution of airway mucous metaplasia.

Exacerbations of muco-obstructive airway diseases such as COPD and asthma are associated with epithelial changes termed mucous metaplasia (MM). Many molecular pathways triggering MM have been identified; however, the factors that regulate resolution are less well understood. We hypothesized that the autophagy pathway is required for resolution of MM by eliminating excess non-secreted intracellular mucin granules. We found increased intracellular levels of mucins Muc5ac and Muc5b in mice deficient in autophagy regulatory protein, Atg16L1, and that this difference was not due to defects in the known baseline or stimulated mucin secretion pathways. Instead, we found that, in mucous secretory cells, Lc3/Lamp1 vesicles colocalized with mucin granules particularly adjacent to the nucleus, suggesting that some granules were being eliminated in the autophagy pathway rather than secreted. Using a mouse model of MM resolution, we found increased lysosomal proteolytic activity that peaked in the days after mucin production began to decline. In purified lysosomal fractions, Atg16L1-deficient mice had reduced proteolytic degradation of Lc3 and Sqstm1 and persistent accumulation of mucin granules associated with impaired resolution of mucous metaplasia. In normal and COPD derived human airway epithelial cells (AECs), activation of autophagy by mTOR inhibition led to a reduction of intracellular mucin granules in AECs. Our findings indicate that during peak and resolution phases of MM, autophagy activity rather than secretion is required for elimination of some remaining mucin granules. Manipulation of autophagy activation offers a therapeutic target to speed resolution of MM in airway disease exacerbations.

New perspectives regarding ß(2) -adrenoceptor ligands in the treatment of asthma.

In the last two decades several significant changes have been proposed in the receptor theory that describes how ligands can interact with G protein-coupled receptors (GPCRs). Here we briefly summarize the evolution of receptor theory and detail recent prominent advances. These include: (i) the existence of spontaneously active GPCRs that are capable of signalling even though they are unoccupied by any ligand; (ii) the discovery of ligands that can inactivate these spontaneously active receptors; (iii) the notion that a ligand may simultaneously activate more than one GPCR signalling pathway; and (iv) the notion that certain ligands may be able to preferentially direct receptor signalling to a specific pathway. Because the data supporting these receptor theory ideas are derived primarily from studies using artificial expression systems, the physiological relevance of these new paradigms remains in question. As a potential example of how these new perspectives in receptor theory relate to drug actions and clinical outcomes, we discuss their relevance to the recent controversy regarding the chronic use of ß(2) -adrenoceptor agonists in the treatment of asthma.

Inhaled corticosteroids stabilize constrictive bronchiolitis after hematopoietic stem cell transplantation.

Post transplantation constrictive bronchiolitis (PTCB) is the most common pulmonary complication among long-term survivors of allogeneic hematopoietic stem cell transplantation (HSCT). It is a late manifestation of GVHD. Its treatment with high-dose systemic corticosteroids and other immunosuppressive regimens is associated with multiple side effects. Topical corticosteroids are used for the treatment of other manifestations of GVHD to minimize these side effects. We conducted a retrospective analysis of a series of adult patients to evaluate the efficacy of high-dose inhaled corticosteroids in the treatment of PTCB. Seventeen patients with new-onset airflow obstruction were diagnosed with PTCB. Their forced expiratory volume in 1 s (FEV1) declined from a median of 84% (range, 56-119) before HSCT to 53% (26-82) after HSCT. All patients received inhaled fluticasone propionate 500-940 microg two times daily. Symptoms of airway obstruction improved and FEV1 stabilized 3-6 months after treatment. We conclude that high-dose inhaled corticosteroids may be effective in the treatment of PTCB and propose a plausible mechanism of its action. A prospective evaluation of its efficacy is warranted.

Outcome of alveolar hemorrhage in hematopoietic stem cell transplant recipients.

Alveolar hemorrhage (AH) is a frequent, serious complication of hematopoietic stem cell transplantation (HSCT). To study the incidence of AH, its clinical course and outcomes in HSCT patients, a retrospective review of the records of all adult patients who underwent bronchoscopy between January 1, 2002 and December 31, 2004 was carried out and those who underwent bronchoscopy after HSCT identified. A total of 223 patients underwent bronchoscopy after HSCT for diffuse pulmonary infiltrates with respiratory compromise. Eighty-seven (39%) patients had AH. Of these, 53 had AH without any identified organism while 34 had an organism along with hemorrhage on bronchoalveolar lavage (BAL). Six-month survival rate of patients with AH was 38% (95% confidence interval: 27-48%). In 95 of the 223 patients, an organism was isolated from BAL. These patients had poor outcomes compared to patients in whom no organism was identified. Patients with both AH and an organism had the worst prognosis. Mortality of patients with AH is improving and long-term survival of patients with AH is feasible. Isolation of a microbial organism in BAL is a strong predictor of poor outcome.

Stenotrophomonas maltophilia pneumonia in cancer patients without traditional risk factors for infection, 1997-2004.

In order to elucidate the spectrum of Stenotrophomonas maltophilia pneumonia in cancer patients without traditional risk factors, 44 cancer patients (cases) with S. maltophilia pneumonia in whom S. maltophilia pneumonia risk factors were not present were compared with two S. maltophilia pneumonia risk groups (controls) including 43 neutropenic non-intensive care unit (ICU) and 21 non-neutropenic ICU patients. The case and control patients had similar demographic and underlying clinical characteristics. Compared with case patients with S. maltophilia pneumonia, neutropenic patients had higher exposure to carbapenem antibiotics (58 vs. 41%; p < 0.03), more frequent hematologic malignancy (95 vs. 64%; p < 0.0003), and they presented with concurrent bacteremia more often (23 vs. 0%; p < 0.0005). Patients with S. maltophilia pneumonia in the ICU needed vasopressor therapy more frequently than cases (62 vs. 5%; p < 0.0001). Hospital-acquired S. maltophilia pneumonia was more common among controls than cases (98 vs. 61%; p < 0.000002). Among the cases, 15 (34%) received outpatient oral antimicrobial therapy, while 29 were hospitalized and eight (28%) were subsequently admitted to the ICU. The mean duration of ICU stay, even among these eight patients (19 +/- 40 days), was comparable to that of patients with neutropenia (23 +/- 26 days) and those who developed S. maltophilia pneumonia during their ICU stay (34 +/- 22 days; p = 0.46). The overall infection-associated mortality in the 108 patients with S. maltophilia pneumonia was 25%. Twenty percent of patients without traditional risk factors for S. maltophilia pneumonia died due to progressive infection. In a multivariate logistic regression analysis, only admission to the ICU predicted death (odds ratio 33; 95% confidence interval, 4.51-241.2; p < 0.0006). The results of this study indicate S. maltophilia pneumonia is a serious infection even in non-neutropenic, non-ICU patients with cancer.

Inhibition of mucin secretion with MARCKS-related peptide improves airway obstruction in a mouse model of asthma.

Allergic asthma is associated with airway epithelial cell mucous metaplasia and mucin hypersecretion, but the consequences of mucin hypersecretion on airway function are unclear. Recently, a peptide derived from the myristoylated alanine-rich C kinase substrate protein NH(2)-terminal sequence (MANS) was shown to inhibit methacholine (MCh)-induced mucin secretion from airway mucous cells by >90%. We studied the effect of intranasal pretreatment with this peptide on specific airway conductance (sGaw) during challenge with MCh in mice with allergen-induced mucous cell metaplasia. sGaw was noninvasively measured in spontaneously breathing restrained mice, using a double-chamber plethysmograph. Pretreatment with MANS peptide, but not a control peptide [random NH(2)-terminal sequence (RNS)], resulted in partial inhibition of the fall in sGaw induced by 60 mM MCh (mean +/- SE; baseline 1.15 +/- 0.06; MANS/MCh 0.82 +/- 0.05; RNS/MCh 0.55 +/- 0.05 cmH(2)O/s). The protective effect of MANS was also seen in mice challenged with allergen for 3 consecutive days to increase airway hyperresponsiveness, although the degree of protection was less (baseline 1.1 +/- 0.08; MANS/MCh, 0.65 +/- 0.06; RNS/MCh 0.47 +/- 0.03 cmH(2)O/s). Because routine sGaw measurement in mice includes nasal airways, the effectiveness of MANS was also confirmed in mice breathing through their mouths after nasal occlusion (baseline 0.92 +/- 0.05; MANS/MCh 0.83 +/- 0.06; RNS/MCh 0.61 +/- 0.03 cmH(2)O/s). In all instances, sGaw in the MANS-pretreated group was approximately 35% higher than in RNS-treated controls, and mucous obstruction accounted for approximately 50% of the MCh-induced fall in sGaw. In summary, mucin secretion has a significant role in airway obstruction in a mouse model of allergic asthma, and strategies to inhibit mucin secretion merit further investigation.

Traffic control: Rab GTPases and the regulation of interorganellar transport.

Membrane proteins, membrane lipids, and luminal contents are exchanged among the intracellular compartments of eukaryotic cells by vesicular transport. This process must be highly ordered to maintain cellular architecture in the face of rapid membrane turnover. The Ras-related Rab GTPases play multiple roles in regulating this traffic.

Gene structure and promoter function of murine Munc18-2, a nonneuronal exocytic Sec1 homolog.

Sec1 family proteins are regulators of diverse exocytic processes, from yeast to man. Three mammalian homologues, Munc18-1, -2, and -3 have been described. We have studied the structure and expression of the mouse Munc18-2 gene. The Munc18-2 gene comprises 19 exons whose sizes range from 50 to 158 bp, with a total gene size of approximately 11 kb. A single transcript of 2.1 kb is expressed in multiple non-neuronal murine tissues. Munc18-2 has a striking resemblance to Munc18-1 in structure despite only 60% sequence identity, suggesting a recent gene duplication event. Analysis of the region upstream of the transcription start site shows that Munc18-2 has a TATA-less promoter, with a consensus initiator (Inr) sequence at the start of transcription, several Sp1 binding sites, and strong promoter activity in RBL-2H3 mast cells. The region from +5 to -430 is more active than +5 to -800, suggesting upstream repressor elements.

Genomic organization, chromosomal localization, and expression of the murine RAB3D gene.

Rab proteins, members of the Ras superfamily of small GTPases, play regulatory roles in intercompartmental vesicular transport. Each step of traffic seems to require the participation of at least one distinct Rab, with the Rab3 subfamily involved in stimulated exocytosis. We report our studies on the murine rab3D gene, one of the four mammalian Rab3 isoforms. We located this gene on chromosome 13, region A(2-3). The rab3D gene consists of 5 exons spanning 10.6 kb, and the structural gene is contained in exons 2 through 5 with one canonical GTP-binding motif in each exon. Organization of the rab3D gene is identical to that of rab3A but different from other rab genes. Alternative poly-A(+) signals in the 3' untranslated region account for the identities of multiple transcripts detected by Northern blot analysis. Rab3D is expressed in all tissues studied, predominantly in heart, lung, and liver, and binding sites for multiple transcription factors are found in the TATA-less promoter region.

A novel membrane-anchored Rab5 interacting protein required for homotypic endosome fusion.

The ras-related GTPase rab5 is rate-limiting for homotypic early endosome fusion. We used a yeast two-hybrid screen to identify a rab5 interacting protein, rab5ip. The cDNA sequence encodes a ubiquitous 75-kDa protein with an N-terminal transmembrane domain (TM), a central coiled-coil structure, and a C-terminal region homologous to several centrosome-associated proteins. rab5ip lacking the transmembrane domain (rab5ipTM(-)) had a greater affinity in vitro for rab5-guanosine 5'-O-2-(thio)diphosphate than for rab5-guanosine 5'-3-O-(thio)triphosphate. In transfected HeLa cells, rab5ipTM(-) was partly cytosolic and localized (by immunofluorescence) with a rab5 mutant believed to be in a GDP conformation (GFP-rab5(G78A)) but not with GFP-rab5(Q79L), a GTPase-deficient mutant. rab5ip with the transmembrane domain (rab5ipTM(+)) was completely associated with the particulate fraction and localized extensively with GFP-rab5(wt) in punctate endosome-like structures. Overexpression of rab5ipTM(+) using Sindbis virus stimulated the accumulation of fluid-phase horseradish peroxidase by BHK-21 cells, and homotypic endosome fusion in vitro was inhibited by antibody against rab5ip. rab5ipTM(-) inhibited rab5(wt)-stimulated endosome fusion but did not inhibit fusion stimulated by rab5(Q79L). rab5ip represents a novel rab5 interacting protein that may function on endocytic vesicles as a receptor for rab5-GDP and participate in the activation of rab5.

Synaptotagmin II negatively regulates Ca2+-triggered exocytosis of lysosomes in mast cells.

Synaptotagmins (Syts) I and II are believed to act as Ca2+ sensors in the control of neurotransmission. Here we demonstrate that mast cells express Syt II in their lysosomal fraction. We further show that activation of mast cells by either aggregation of FcepsilonRI or by Ca2+ ionophores results in exocytosis of lysosomes, in addition to the well documented exocytosis of their secretory granules. Syt II directly regulates lysosomal exocytosis, whereby overexpression of Syt II inhibited Ca2+-triggered release of the lysosomal processed form of cathepsin D, whereas suppression of Syt II expression markedly potentiated this release. These findings provide evidence for a novel function of Syt II in negatively regulating Ca2+-triggered exocytosis of lysosomes, and suggest that Syt II-regulated secretion from lysosomes may play an important role in mast cell biology.

Agonist-induced sorting of human beta2-adrenergic receptors to lysosomes during downregulation.

During prolonged exposure to agonist, beta2-adrenergic receptors undergo downregulation, defined by the loss of radioligand binding sites. To determine the cellular basis for beta2-adrenergic receptor downregulation, we examined HEK293 cells stably expressing beta2-adrenergic receptors with an N-terminal epitope tag. Downregulation was blocked by leupeptin, a cysteine protease inhibitor, but not by pepstatin, an inhibitor of aspartate proteases. Immunofluorescence microscopy of cells treated with agonist for 3-6 hours in the presence of leupeptin showed beta2-adrenergic receptors, but not transferrin receptors, localizing with the lysosomal protease cathepsin D, and with lysosomes labeled by uptake of a fluorescent fluid-phase marker. No localization of beta2-adrenergic receptors with lysosomal markers was observed in the absence of leupeptin, most likely due to proteolysis of the epitope. The proton pump inhibitor, bafilomycin A1, significantly inhibited this agonist-induced redistribution of beta2-adrenergic receptors into lysosomes, causing receptors to accumulate in the rab11-positive perinuclear recycling compartment and slowing the rate of beta2-adrenergic receptor recycling. Control experiments showed that leupeptin had no nonspecific effects on the cellular trafficking of either beta2-adrenergic receptors or transferrin receptors. Although cAMP alone caused a small decline in receptor levels without redistributing beta2-adrenergic receptors from the plasma membrane, this effect was additive to that seen with agonist alone, suggesting that agonist-induced beta2-adrenergic receptor downregulation resulted largely from cAMP-independent mechanisms. These results indicate that during agonist-induced downregulation, a significant fraction of beta2-adrenergic receptors are specifically sorted to lysosomes via the endosomal pathway, where receptor degradation by cysteine proteases occurs. These results provide a cellular explanation for the loss of radioligand binding sites that occurs during prolonged exposure to agonist.

Rab3D, a small GTPase, is localized on mast cell secretory granules and translocates to the plasma membrane upon exocytosis.

Although mast cell secretion has been intensively studied because of its pivotal role in allergic reactions and its advantages as a physiologic model, the molecular composition of the secretory machine is virtually unknown. In view of the guanine-nucleotide dependency of mast cell exocytosis and the participation of Rab3 proteins in synaptic vesicle release, we hypothesized that a Rab3 isoform regulates mast cell secretion. Fragments of Rab3A, 3B, and 3D were cloned from RBL-2H3 mast cells by reverse transcription- polymerase chain reaction (RT-PCR). Northern blot analysis revealed Rab3D transcripts to be relatively abundant, Rab3B substantially less so, and Rab3A and 3C undetectable. By ribonuclease (RNase) protection assay, Rab3D transcripts were at least 10-fold more abundant than those of other isoforms, and by immunoblot analysis, Rab3D protein was at least 60-fold more abundant than that of Rab3B. Rab3D was more abundant in RBL cells than in brain, but the total mass of Rab3 proteins in RBL cells was 10-fold less than in brain. Rab3D only partly colocalized with secretory granules in RBL cells, but fully colocalized in mature peritoneal mast cells. There was a descending concentration gradient of Rab3D from peripheral to central granules, and no cytoplasmic pool was detectable in resting mast cells. Following exocytotic degranulation, Rab3D translocated to the plasma membrane and remained there for at least 15 min. These studies suggest that Rab3D is a component of the regulated exocytotic machine of mast cells, and identify differences between mast cells and neurons in Rab3 expression and trafficking.

Conformationally variable Rab protein surface regions mapped by limited proteolysis and homology modelling.

Tryptic proteolysis of the small GTPases Rab4 and Rab5 is a multi-step, nucleotide-dependent process. Using N-terminal peptide sequencing, matrix-assisted laser desorption ionization-time-of-flight MS and molecular modelling, we identified the three initial sites of proteolysis in Rab5 as Arg-4, Arg-81 and Arg-197. Arg-4 and Arg-81 lie within regions previously implicated in Rab5 endocytic function, and Arg-197 lies in a region involved in membrane targeting. Topologically, Arg-81 lies within the conformationally variable Switch II region shown to be important for protein-protein interactions of other GTPases. Homology modelling studies on Rab5 indicate that the Arg-81 side chain is buried in the Rab5 GTP conformation, but is solvent-accessible in the GDP conformation, explaining the dependence of proteolysis on nucleotides. Peptide mapping of Rab4 was performed to take advantage of additional scissile bonds within Switch II to determine more precisely the limits of the nucleotide-dependent protease-accessible region. The Rab4 cleavage sites corresponded to Arg-81 and Pro-87 of Rab5, and taken together with the finding that Rab5 was not cleaved at Arg-91 this analysis defines an eight-residue surface-exposed conformationally variable region lying in the centre of Switch II. A sequence comparison of Rab proteins shows these eight residues to have a loosely conserved motif that we term Switch II(v) for its relative variability. C-terminal to Switch II(v) is a highly conserved Rab-specific YYRGA motif that we term Switch II(c) for its constant sequence. N-terminal to Switch II(v) is a sequence-invariant G-domain involved in nucleotide binding and hydrolysis. We propose that the Rab Switch II(v) region imparts specificity to nucleotide-dependent protein-protein interactions.

Evidence for a symmetrical requirement for Rab5-GTP in in vitro endosome-endosome fusion.

Early endosome fusion, which has been extensively characterized using an in vitro reconstitution assay, is Rab5-dependent. To examine the requirement for Rab5 on both fusion partners, we prepared cytosol and endosomes depleted of Rab5. Unlike control cytosol, Rab5-depleted cytosol was only marginally active in the in vitro endosome fusion. However, fusion could be restored by the addition of wild-type Rab5 or Rab5 D136N, a mutant whose nucleotide specificity favors xanthine over guanine. The addition of Rab5 D136N restored fusion only in the presence of XTP. In the absence of XTP or in the presence of XDP, Rab5 D136N failed to restore fusion. When fusion was carried out with endosomal vesicles depleted of Rab GTPases (by preincubation of vesicles with GDP dissociation inhibitor), together with cytosol immunodepleted of Rab5, fusion was virtually absent. We then used immunodepleted cytosol and GDP dissociation inhibitor-treated vesicles to determine whether Rab5 is required by both fusion partners. Using separate sets of endosomal vesicles, we found that priming both sets of Rab5-depleted vesicles with Rab5 Q79L, a GTPase-defective mutant, substantially stimulated endosome fusion. Priming one set of vesicles with Rab5 Q79L and a second set of vesicles with Rab5 S34N failed to activate fusion. When both sets of Rab5-depleted vesicles were primed with Rab5 D136N supplemented with XTP, endosome fusion was stimulated, similar to that observed with Rab5 Q79L. However, when one set of vesicles was preincubated with Rab5 D136N plus XTP and the second set with Rab5 D136N and XDP, no stimulation of fusion was observed. We conclude that Rab5-GTP is required on both fusion partners for docking and fusion of early endosomes. To confirm the fusion of Rab5-GTP-positive vesicles in vivo, we expressed GFP-Rab5 Q79L in fibroblasts and observed fusion of Rab5-positive vesicles. We failed to record fusion of Rab5-positive vesicles with Rab5-negative vesicles. We conclude that Rab5-GTP is required on both sets of endosomes for fusion in vitro and in living cells.

Atrial natriuretic peptide modulates alveolar type 2 cell adenylyl and guanylyl cyclases and inhibits surfactant secretion.

Alveolar epithelial type 2 (T2) cells isolated from the lungs of adult rats responded to exogenous atrial natriuretic peptide (ANP) by two signalling mechanisms. First, ANP induced a dose-dependent reduction of ligand-stimulated adenylyl cyclase activity and cAMP accumulation. This effect was inhibited by the addition of GDPbetaS or by pretreatment with pertussis toxin (PT), consistent with mediation by a Gi protein(s). PT-catalyzed [32P]ADP-ribosylation, immunoblots with specific antisera, and Northern blot analysis demonstrated that T2 cells contain the G-proteins Gi2 and Gi3 which could transduce this signal. ANP also promoted PT-insensitive, dose-dependent accumulation of cGMP, consistent with activation of a receptor guanylyl cyclase. Isoproterenol-stimulated phosphatidylcholine secretion was markedly attenuated by ANP, and this effect was inhibited by PT pretreatment, consistent with mediation by a Gi protein(s). These data indicate that in addition to the lung being a major clearance organ for circulating ANP, lung parenchymal cells are targets of ANP action.

Long-acting inhaled beta2-agonists in asthma therapy.

OBJECTIVE: To review the pharmacology of the long-acting inhaled beta2-agonists, salmeterol and formoterol, summarize results of their clinical trials, evaluate their safety records, and discuss their roles in the treatment of asthma. DATA SOURCES: Preclinical and clinical studies involving salmeterol or formoterol were identified by a MEDLINE search, weekly computerized literature updates, and manual searches. Studies of satisfactory quality were chosen for review. DATA SYNTHESIS: Salmeterol and formoterol are potent and selective beta2-adrenoceptor agonists with durations of action >12 h. Their major differences are that formoterol has a rapid onset of action and is a partial agonist of high intrinsic efficacy, whereas salmeterol has a delayed onset and is a partial agonist of low intrinsic efficacy. Twice daily use of either drug results in improved lung function, reduced symptoms, and a better quality of life. These agents protect against exercise-induced asthma for 12 h and eliminate nighttime awakening in most patients. Limited tolerance develops, especially to their bronchoprotective effects, but their improvement of lung function is sustained. CONCLUSIONS: Regular use of salmeterol or formoterol provides subjective and objective amelioration of asthma in patients experiencing excessive symptoms or physiologic impairment despite the regular administration of low doses of inhaled corticosteroids (equivalent to approximately 500 microg/d of beclomethasone). Intermittent use of either long-acting beta2-agonist can provide prolonged protection against exercise-induced asthma or nighttime symptoms. Patients should be instructed to continue taking inhaled steroids when long-acting beta2-agonists are administered on a regular schedule and to not take long-acting beta2-agonists between regularly scheduled doses. Used properly, they are effective and safe adjunctive agents in the treatment of asthma.