Physical characterization of a low-charge glycoform of the MUC5B mucin comprising the gel-phase of an asthmatic respiratory mucous plug.
We have previously noted that sequential extraction of an asthmatic mucous exudate with 6 M guanidinium chloride yielded a fraction of the mucins that were most resistant to solubilization and of high Mr [Sheehan, Richardson, Fung, Howard and Thornton (1995) Am. J. Respir. Cell Mol. Biol. 13, 748-756]. Here we show that this mucin fraction is dominated (at least 96% of the total) by the low-charge glycoform of the MUC5B gene product. Seen in the electron microscope the mucins appeared mainly as compact 'island' structures composed of linear threads often emanating from globular 'nodes' rather than the discrete linear threads more typical of mucins that we have previously described. The effect of reducing agents was as expected for other gel-forming mucins, i.e. reduced subunits or monomers of Mr 3x10(6)) were produced within 15 min of treatment. Kinetic experiments on the cleavage of the intact mucins with the proteinase trypsin indicated two clear regimes of fragmentation. An initial rapid cleavage generated mucins ranging from Mr=4x10(6) to 30x10(6) that in the electron microscope appeared as polydisperse threads (500-3000 nm in length), similar to normal and other respiratory mucins that we have previously characterized. A subsequent slower fragmentation over many hours yielded a major fragment of Mr 3x10(6) and length 200-600 nm, very similar in size and Mr to the subunits obtained by reduction. The results suggest that the MUC5B mucin is assembled, first into polydisperse linear threads, which are then linked together via a protein-mediated process. This might involve part of the mucin polypeptide or an as yet unidentified protein(s). The high proteinase susceptibility of the linkage suggests that it might be a point of control for mucin size and thus mucus rheology. (+info)
Developmental changes in mucosubstances revealed by immunostaining with antimucus monoclonal antibodies and lectin staining in the epithelium lining the segment from gizzard to duodenum of the chick embryo.
The mucosubstances in the epithelium lining the segment from gizzard to duodenum during development of the chick embryo was studied histochemically using monoclonal antibodies against gizzard mucus and lectins, with attention to the regional differentiation of the epithelium in this segment. The anterior limit of epithelial CdxA mRNA expression detected by in situ hybridisation, which served as the position of the gizzard-duodenal boundary, was clearly found from d 3. Granules positive for some antibodies or lectins were found in the region ranging from the posterior part of the gizzard to the duodenum at d 3, which was followed by an increase in the number of granules and a gradual enlargement of the granule-positive area to the anterior part of the gizzard over 4-6 d. From d 4, the epithelia of the gizzard body and of the pyloric or duodenal region came to be differently stained with some antibodies or lectins. From d 10, each region showed a specific pattern of staining. The epithelia of the gizzard body and pyloric region contained abundant mucus granules with a different staining pattern. In the duodenum the number of stained granules was low except in occasional goblet cells. Thus the epithelia of the gizzard body, pyloric region and duodenum may produce different mucosubstances and the regional differentiation in these epithelia may start at rather early stages soon after the formation of digestive tube. (+info)
Immunoglobulin-specific radioimmunoprecipitation assays for quantitation of nasal secretory antibodies to hemagglutinin of type A influenza viruses.
Radioimmunoprecipitation (RIP) assays were developed to selectively quantitate class-specific antibodies to purified hemagglutinins (HA) of type A influenza virus in nasal secretions. Rabbit anti-human secretory piece of immunoglobulin A (IgA) and rabbit anti-human IgG were used as second antibodies. A third antibody, goat anti-rabbit IgG, was incorporated into the system to separate immune complexes formed between iodinated HA, nasal wash test specimen, and second antibody. The utilization of this reagent avoided the need for large quantities of IgA and IgG antibody-negative carrier secretions. Nasal was specimens obtained from 14 adults immunized with an inactivated type A influenza virus vaccine were evaluated by RIP and viral neutralization assays. Significant homologous postvaccination secretory IgA and IgG antibody levels were demonstrable in 13 (93%) of individuals by RIP, whereas only 5 (36%) exhibited rises by viral neutralization tests. Moreover, the geometric mean IgA and IgG antibody levels were at least 20- and 37-fold greater than the neutralizing antibody titer. The pattern of heterologous immunoglobulin-specific antibody responses tended to be similar to those observed with the homologous HA subunit. (+info)
Neuroregulation by vasoactive intestinal peptide (VIP) of mucus secretion in ferret trachea: activation of BK(Ca) channels and inhibition of neurotransmitter release.
1. The aims of this study were to determine: (1) whether vasoactive intestinal peptide (VIP) regulates cholinergic and 'sensory-efferent' (tachykininergic) 35SO4 labelled mucus output in ferret trachea in vitro, using a VIP antibody, (2) the class of potassium (K+) channel involved in VIP-regulation of cholinergic neural secretion using glibenclamide (an ATP-sensitive K+ (K(ATP)) channel inhibitor), iberiotoxin (a large conductance calcium activated K+ (BK(ca)) channel blocker), and apamin (a small conductance K(ca) (SK(ca)) channel blocker), and (3) the effect of VIP on cholinergic neurotransmission using [3H]-choline overflow as a marker for acetylcholine (ACh) release. 2. Exogenous VIP (1 and 10 microM) alone increased 35SO4 output by up to 53% above baseline, but suppressed (by up to 80% at 1 microM) cholinergic and tachykininergic neural secretion without altering secretion induced by ACh or substance P (1 microM each). Endogenous VIP accounted for the minor increase in non-adrenergic, non-cholinergic (NANC), non-tachykininergic neural secretion, which was compatible with the secretory response of exogenous VIP. 3. Iberiotoxin (3 microM), but not apamin (1 microM) or glibenclamide (0.1 microM), reversed the inhibition by VIP (10 nM) of cholinergic neural secretion. 4. Both endogenous VIP (by use of the VIP antibody; 1:500 dilution) and exogenous VIP (0.1 microM), the latter by 34%, inhibited ACh release from cholinergic nerve terminals and this suppression was completely reversed by iberiotoxin (0.1 microM). 5. We conclude that, in ferret trachea in vitro, endogenous VIP has dual activity whereby its small direct stimulatory action on mucus secretion is secondary to its marked regulation of cholinergic and tachykininergic neurogenic mucus secretion. Regulation is via inhibition of neurotransmitter release, consequent upon opening of BK(Ca) channels. In the context of neurogenic mucus secretion, we propose that VIP joins NO as a neurotransmitter of i-NANC nerves in ferret trachea. (+info)
Whirling disease: host specificity and interaction between the actinosporean stage of Myxobolus cerebralis and rainbow trout Oncorhynchus mykiss.
Scanning electron microscopic studies were conducted on rainbow trout Oncorhynchus mykiss in the first 60 min after their exposure to the triactinomyxon spores of Myxobolus cerebralis. The results demonstrated that as early as 1 min post exposure the whole process, from the attachment of the triactinomyxon spores to the complete penetration of their sporoplasm germs, had occurred. The triactinomyxon spores sought out the secretory openings of mucous cells of the epidermis, the respiratory epithelium and the buccal cavity of trout and used them as portals of entry. Exposure experiments of the triactinomyxon spores of M. cerebralis to non-salmonid fish, such as goldfish Carassius auratus, carp Cyprinus carpio, nose Chondrostoma nasus, medaka Oryzias latipes, guppy Poecilia reticulata and also the amphibian tadpole Rana pipiens as well as to rainbow trout fry indicated a specificity for salmonids. Attempts to activate the triactinomyxon spores by exposure to mucus prepared from cyprinid and salmonid fish showed no significant differences from those conducted in tap water. The results suggest that the simultaneous presence of both mechano- and chemotactic stimuli was required for finding the salmonid fish host. (+info)
Pulmonary expression of interleukin-13 causes inflammation, mucus hypersecretion, subepithelial fibrosis, physiologic abnormalities, and eotaxin production.
Interleukin (IL)-13 is a pleiotropic cytokine produced in large quantities by activated CD4(+) Th2 lymphocytes. To define further its potential in vivo effector functions, the Clara cell 10-kDa protein promoter was used to express IL-13 selectively in the lung, and the phenotype of the resulting transgenic mice was characterized. In contrast to transgene-negative littermates, the lungs of transgene-positive mice contained an inflammatory response around small and large airways and in the surrounding parenchyma. It was mononuclear in nature and contained significant numbers of eosinophils and enlarged and occasionally multinucleated macrophages. Airway epithelial cell hypertrophy, mucus cell metaplasia, the hyperproduction of neutral and acidic mucus, the deposition of Charcot-Leyden-like crystals, and subepithelial airway fibrosis were also prominently noted. Eotaxin protein and mRNA were also present in large quantities in the lungs of the transgene-positive, but not the transgene-negative, mice. IL-4, IL-5, granulocyte-macrophage colony-stimulating factor, and monocyte chemoattractant protein-5 were not similarly detected. Physiological evaluations revealed significant increases in baseline airways resistance and airways hyperresponsiveness (AHR) to methacholine in transgene-positive animals. Thus, the targeted pulmonary expression of IL-13 causes a mononuclear and eosinophilic inflammatory response, mucus cell metaplasia, the deposition of Charcot-Leyden-like crystals, airway fibrosis, eotaxin production, airways obstruction, and nonspecific AHR. IL-13 may play an important role in the pathogenesis of similar responses in asthma or other Th2-polarized tissue responses. (+info)
Pneumococcus activation of the 5-lipoxygenase pathway and production of glycoproteins in the middle ear of rats.
Pneumococcal otitis media is associated with the production of potent inflammatory mediators (leukotrienes), but the mechanism by which pneumococcus induces production of leukotrienes in the middle ear is poorly understood. In this study, up-regulation of 2 genes that govern the lipoxygenase pathway, cPLA2 and 5-LOX, was observed in rats following inoculation of pneumococcus into the middle ear cavity. Expression of cPLA2 was low, and 5-LOX gene expression was not detected in control animals. Up-regulation of cPLA2 and 5-LOX in middle ear epithelial cells was accompanied by an increase of high-molecular-weight glycoproteins in middle ear fluid and cells. These findings suggest that pneumococcus activates the lipoxygenase pathway by up-regulating expression of the cPLA2 and 5-LOX genes. This, in turn, may stimulate synthesis and secretion of high-molecular-weight glycoproteins that facilitate production of fluid in the middle ear cleft. (+info)
Regulation of 15-lipoxygenase expression and mucus secretion by IL-4 in human bronchial epithelial cells.
Our laboratory has recently shown that mucus differentiation of cultured normal human tracheobronchial epithelial (NHTBE) cells is accompanied by the increased expression of 15-lipoxygenase (15-LO). We used differentiated NHTBE cells to investigate the regulation of 15-LO expression and mucus secretion by inflammatory cytokines. Interleukin (IL)-4 and IL-13 dramatically enhanced the expression of 15-LO, whereas tumor necrosis factor-alpha, IL-1beta, and interferon (IFN)-gamma had no effect. These cytokines did not increase the expression of cyclooxygenase-2, with the exception of a modest induction by IL-1beta. The IL-4-induced 15-LO expression was concentration dependent, and mRNA and protein expression increased within 3 and 6 h, respectively, after IL-4 treatment. In metabolism studies with intact cells, 15-hydroxyeicosatetraenoic acid (15-HETE) and 13-hydroxyoctadecadienoic acid (13-HODE) were the major metabolites formed from exogenous arachidonic acid and linoleic acid. No prostaglandins were detected. IL-4 treatment dramatically increased the formation of 13-HODE and 15-HETE compared with that in untreated NHTBE cells, and several additional 15-LO metabolites were observed. Pretreatment of NHTBE cells with IFN-gamma or dexamethasone did not inhibit the IL-4-induced expression of 15-LO except at high concentrations (100 ng/ml of IFN-gamma and 10 microM dexamethasone). IL-4 treatment inhibited mucus secretion and attenuated the expression of the mucin genes MUC5AC and MUC5B at 12-24 h after treatment. Addition of 15-HETE precursor and 13-HODE precursor to the cultures did not alter mucin secretion or mucin gene expression. On the basis of the data presented, we conclude that the increase in 15-LO expression by IL-4 and attenuation of mucus secretion may be independent biological events. (+info)