Mucus Secretagogue Research Articles

IgGFc-binding protein and MUC2 mucin produced by colonic goblet-like cells spatially interact non-covalently and regulate wound healing.

The colonic mucus bilayer is the first line of innate host defense that at the same time houses and nourishes the commensal microbiota. The major components of mucus secreted by goblet cells are MUC2 mucin and the mucus-associated protein, FCGBP (IgGFc-binding protein). In this study, we determine if FCGBP and MUC2 mucin were biosynthesized and interacted together to spatially enhance the structural integrity of secreted mucus and its role in epithelial barrier function. MUC2 and FCGBP were coordinately regulated temporally in goblet-like cells and in response to a mucus secretagogue but not in CRISPR-Cas9 gene-edited MUC2 KO cells. Whereas ~85% of MUC2 was colocalized with FCGBP in mucin granules, ~50% of FCGBP was diffusely distributed in the cytoplasm of goblet-like cells. STRING-db v11 analysis of the mucin granule proteome revealed no protein-protein interaction between MUC2 and FCGBP. However, FCGBP interacted with other mucus-associated proteins. FCGBP and MUC2 interacted via N-linked glycans and were non-covalently bound in secreted mucus with cleaved low molecular weight FCGBP fragments. In MUC2 KO, cytoplasmic FCGBP was significantly increased and diffusely distributed in wounded cells that healed by enhanced proliferation and migration within 2 days, whereas, in WT cells, MUC2 and FCGBP were highly polarized at the wound margin which impeded wound closure by 6 days. In DSS colitis, restitution and healed lesions in Muc2+/+ but not Muc2-/- littermates, were accompanied by a rapid increase in Fcgbp mRNA and delayed protein expression at 12- and 15-days post DSS, implicating a potential novel endogenous protective role for FCGBP in wound healing to maintain epithelial barrier function.

Colonic Goblet Cells Produce Augmented Levels of Pro‐inflammatory Cytokines due to Metabolically Stressful MUC2 Mucin Biosynthesis

An intact mucus layer is vital in innate host defense in the gastrointestinal tract to protect the underlying mucosal epithelium from potential pathogens and toxins from the external environment whilst supporting the commensal microbiota. In the colon, goblet cells produce this essential mucus bilayer by secreting MUC2 mucin under a highly ER stressful mechanism. In conditions such as inflammatory bowel diseases and infectious colitis, goblet cells hypersecrete mucins resulting in depletion that disrupts the protective mucus layers. Goblet cells also produce pro‐inflammatory cytokines that interact with and activate other arms of the innate immune system in host defence. In this study we hypothesize that ER stress induced by MUC2 biosynthesis and production augment pro‐inflammatory responses in goblet cells. The aims of the study are: 1) to determine the pro‐inflammatory responses in WT and CRISPR/Cas9 MUC2 KO LS174T human goblet cells and 2) to determine the MAPK signalling pathways by which the pro‐inflammatory responses are altered in WT and MUC2 KO goblet cells.mRNA and protein expression of pro‐inflammatory chemokines/cytokines were analyzed via RT‐qPCR and 15‐plex Luminex array, at basal conditions and in response to various mucus secretagogues, including phorbol myristate acetate (PMA, positive control), live Entamoeba histolytica(Eh), and lysed soluble amebic proteins (SAP). To determine the impact of ER stress, the expression of various stress proteins was quantified by Western blotting basally and following treatment with IL‐22 to alleviate ER stress. To determine which MAPK signalling pathways were altered in the cell lines resulting in differing pro‐inflammatory phenotypes, specific pharmacological inhibitors were used and analyzed by Western blotting.WT goblet cells expressed high levels of the ER stress proteins, ATF4 and GRP78, as compared to MUC2 KO cells, both basally and in response to live Eh. In response to the various mucus secretagogues, PMA, live Eh, and SAP, WT cells expressed higher levels of mRNA transcripts and secreted high amounts of the pro‐inflammatory chemokines, interleukin‐8 (IL‐8), interleukin‐13 (IL‐13) and monocyte chemoattractant protein‐1 (MCP‐1) as compared to MUC2 KO cells. Specifically, IL‐8 secretion was significantly elevated in both cell lines by mitigating ER stress with IL‐22 pre‐treatment. In particular, WT cells showed rapid and elevated phospho‐ERK MAPK signalling responses, whereas the response in MUC2 KO cells was delayed.These results demonstrate that goblet cells under high metabolic ER stress triggered by MUC2 mucin biosynthesis activates the ERK MAPK signalling pathway basally and in response to inflammatory agonists to produce elevated levels of pro‐inflammatory chemokines. In contrast, goblet cells that are genetically deleted of MUC2 showed dysregulated pro‐inflammatory responses and altered MAPK signalling that could dysregulate innate host defences analogous to the pathology observed in Muc2‐/‐ littermates.

Pic Protein From Enteroaggregative E. coli Induces Different Mechanisms for Its Dual Activity as a Mucus Secretagogue and a Mucinase.

A hallmark of enteroaggregative Escherichia coli (EAEC) infection is the formation of an intestinal biofilm, which comprises a mucus layer with immersed bacteria. Pic is an autotransporter secreted by EAEC, and other E. coli pathotypes, and has been involved in two apparently contradictory phenotypes, as a mucus secretagogue and as a mucinase. Here, we investigated this Pic dual activity, mucus secretagogue capability and mucinolytic activity, in human goblet cells that secrete MUC2 and MUC5AC. Pic induced mucus hypersecretion directly in the goblet cells, without other intestinal cell types involved. At the same time, Pic exhibited strong proteolytic activity on the secreted mucins. These activities were independent since a mutation in the serine protease motif (PicS258I) abolished mucin degradation while maintaining the mucus secretagogue activity intact. Furthermore, deoxycholic acid (DCA)-induced mucins were proteolytically degraded when goblet cells were co-incubated with DCA/Pic, while co-incubation with DCA/PicS258I induced a synergistic effect on mucus hypersecretion. Pic was more efficient degrading MUC5AC than MUC2, but no degradation was detected with Pic inactivated at the active site by mutation or pharmacological inhibition. Remarkably, Pic cleaved MUC2 and MUC5AC in the C-terminal domain, leaving N-terminal subproducts, impacting the feature of gel-forming mucins and allowing mucus layer penetration by EAEC. Astonishingly, Pic stimulated rapid mucin secretion in goblet-like cells by activating the intracellular calcium pathway resulting from the PLC signal transduction pathway, leading to the production of DAG and releasing IP3, a second messenger of calcium signaling. Therefore, the dual activity of Pic, as a mucus secretagogue and a mucinase, is relevant in the context of carbon source generation and mucus layer penetration, allowing EAEC to live within the layer of mucus but also access epithelial cells.

Role of MUC2 mucin associated FCGBP in innate host defense against Entamoeba histolytica

The mucus layer is the first line of innate host defense in the gastrointestinal tract against potential pathogens and dangerous compounds. The mucus bilayer in the colon is primarily composed of MUC2 mucin, however recent studies have described other proteins secreted with colonic mucus. One of these proteins is FCGBP which is abundantly expressed in mucus and hypothesized to stabilize the mucus layer. This study aims to characterize the biosynthesis and packaging of FCGBP with MUC2 mucin and how it is regulated in response to the colonic parasite Entamoeba histolytica (Eh). Eh cysteine protease 5 (EhCP5) degrades MUC2 mucin in the C terminus to invade the colon. We hypothesize that MUC2 and FCGBP together play important roles in innate host defense. The aims of the study are, 1) to determine if FCGBP co‐localizes with MUC2 mucin and 2) to quantify a functional role for FCGBP with MUC2 against Eh. To elucidate if FCGBP is synthesized and packaged within mucin granule vesicles, granules were isolated from human goblet cells LS174T by differential centrifugation and purified through sucrose gradients. Granules were treated with the detergent CHAPS to solubilize the granule lipid bilayer and the presence of MUC2 and FCGBP determined by Western blotting. To determine if FCGBP mRNA expression was altered in response to Eh, live Eh were incubated with goblet cells for 10, 20, and 30 min and quantified by RT‐PCR. To determine if Eh could degrade FCGBP in a similar fashion as MUC2, secreted proteases from WT Eh and EhCP5‐ were incubated with mucin granules and enzymatic cleavage of FCGBP and MUC2 determined by Western blotting using antibodies specific for the C‐terminus of MUC2 and FCGBP.FCGBP was associated with mucin granules and following treatment with the mild detergent CHAPS, increased immunoreactivity for FCGBP and MUC2 suggesting that FCGBP was localized within granules and not on the granule lipid bilayer. FCGBP partitioned in the same high density fractions as MUC2 granules purified by sucrose density gradient demonstrating that FCGBP was localized with MUC2 within mucin granules. Interestingly, both MUC2 and FCGBP mRNA expressions in goblet cells were co‐ordinately increased in a time‐dependent fashion in response to live Eh and the positive mucus secretagogues, PMA and PGE2. Secreted Eh cysteine protease rapidly degraded both MUC2 and FCGBP as visualized by Western blotting, with FCGBP being completely degraded within 1 min of treatment whereas MUC2 was degraded 50%. Incubation with SPs derived from EhCP5‐ inhibited the degradation of both MUC2 and FCGBP suggesting that degradation of FCGBP occurred prior to MUC2 by destabilizing MUC2.This study demonstrates that FCGBP is localized within MUC2 mucin granules and are secreted together to form an integral part of mucosal innate host defense. Strikingly, both MUC2 and FCGBP mRNA transcripts were co‐ordinately regulated in response to Eh. Importantly, Eh cysteine proteinase rapidly degraded FCGBP prior to cleaving MUC2 in the C‐terminus. Taken together, these data suggest that FCGBP plays an integral role with MUC2 mucin in innate host defense and in conferring rheological properties of the mucus gel in the colon.Support or Funding InformationSupported by CIHR

The delicate balance between Entamoeba histolytica, mucus and microbiota

ABSTRACTEntamoeba histolytica (Eh) is a protozoan parasite of humans that colonizes the outer colonic mucus layer. Under conditions not fully understood, Eh breaches innate host defenses and invades the intestinal mucosa-causing amebic colitis and liver abscess. In asymptomatic infection, Eh interacts with and feeds on resident microbiota that forms biofilms on the outer colonic mucus layer. Despite the close association between Eh and commensal microbiota, we still lack basic knowledge on whether microbiota and/or their metabolites influence Eh virulence traits critical in disease pathogenesis. In the pathogenesis of intestinal amebiasis, Eh overcomes the protective mucus layer using a combination of mucinase/glycosidase and potent mucus secretagogue activity. In this addendum, we discuss the interconnected role of a healthy mucus barrier and the role commensal microbiota play in shaping innate host defense against Eh-induced pro-inflammatory and secretory responses critical in disease pathogenesis.

Colonic levels of vasoactive intestinal peptide decrease during infection and exogenous VIP protects epithelial mitochondria against the negative effects of IFNγ and TNFα induced during Citrobacter rodentium infection

Citrobacter rodentium infection is a model for infection with attaching and effacing pathogens, such as enteropathogenic Escherichia coli. The vasoactive intestinal peptide (VIP) has emerged as an anti-inflammatory agent, documented to inhibit Th1 immune responses and successfully treat animal models of inflammation. VIP is also a mucus secretagogue. Here, we found that colonic levels of VIP decrease during murine C. rodentium infection with a similar time dependency as measurements reflecting mitochondrial function and epithelial integrity. The decrease in VIP appears mainly driven by changes in the cytokine environment, as no changes in VIP levels were detected in infected mice lacking interferon gamma (IFNγ). VIP supplementation alleviated the reduction of activity and levels of mitochondrial respiratory complexes I and IV, mitochondrial phosphorylation capacity, transmembrane potential and ATP generation caused by IFNγ, TNFα and C. rodentium infection, in an in vitro mucosal surface. Similarly, VIP treatment regimens that included the day 5–10 post infection period alleviated decreases in enzyme complexes I and IV, phosphorylation capacity, mitochondrial transmembrane potential and ATP generation as well as increased apoptosis levels during murine infection with C. rodentium. However, VIP treatment failed to alleviate colitis, although there was a tendency to decreased pathogen density in contact with the epithelium and in the spleen. Both in vivo and in vitro, NO generation increased during C. rodentium infection, which was alleviated by VIP. Thus, therapeutic VIP administration to restore the decreased levels during infection had beneficial effects on epithelial cells and their mitochondria, but not on the overall infection outcome.

MUC2 exocytosis in intestinal goblet cells

MUC2 in the major secreted gel‐forming mucin produced by goblet cells (GC) that form the protective mucus blanket covering the epithelium as the first line of innate host defense in the gut. The protein complex and the cellular mechanisms by which MUC2 granulae are released by compound exocytosis from GC under basal or in response to mucus secretagogues are not known. In this study we identified the putative exocytosis components expressed in human GC, LS 174T. The exocytosis protein complex formation between SNAP23, Syntaxin 3 and Munc18b was studied in response to the PKC agonist, phorbol 12‐myristate 13‐acetate (PMA) using immunoprecipitation and mass spectrometry. In contrast to MUC2, the exocytosis proteins were not regulated at the transcriptional level, as mRNA and protein expression was unaltered after PMA. However, PMA treatment phosphorylated Syntaxin 3 in active SNARE complex formation. Surprisingly, confocal imaging revealed that Syntaxin 3 and Munc18b are not only localized to the apical cap of the GC theca but also to areas around the theca where the SNARE components co‐localized with actin filaments. These findings were corroborated using differential centrifugation where Syntaxin 3 was identified on both the granulae and the plasma membrane. Our studies reveal complex exocytosis protein machinery that regulates both basal and stimulated MUC2 granulae release from goblet cells. Supported by CIHR.

Tumor necrosis factor receptor 1-dependent depletion of mucus in immature small intestine: a potential role in neonatal necrotizing enterocolitis.

Necrotizing enterocolitis (NEC) is a leading cause of morbidity and mortality in premature infants. NEC is believed to occur when intestinal bacteria invade the intestinal epithelial layer, causing subsequent inflammation and tissue necrosis. Mucins are produced and secreted by epithelial goblet cells as a key component of the innate immune system and barrier function of the intestinal tract that help protect against bacterial invasion. To better understand the role of mucins in NEC, we quantified the number of mucus-containing small intestinal goblet cells present in infants with NEC and found they had significantly fewer goblet cells and Paneth cells compared with controls. To test whether inflammation has a developmentally dependent effect on intestinal goblet cells, TNF-α was injected into mice at various stages of intestinal development. TNF-α caused a loss of mucus-containing goblet cells only in immature mice and induced Muc2 and Muc3 mRNA upregulation only in mature ileum. Only minimal changes were seen in apoptosis and in expression of markers of goblet cell differentiation. TNF-α increased small intestinal mucus secretion and goblet cell hypersensitivity to prostaglandin E2 (PGE(2)), a known mucus secretagogue produced by macrophages. These TNF-α-induced changes in mucus mRNA levels required TNF receptor 2 (TNFR2), whereas TNF-α-induced loss of mucus-positive goblet cells required TNFR1. Our findings of developmentally dependent TNF-α-induced alterations on intestinal mucus may help explain why NEC is predominantly found in premature infants, and TNF-α-induced alterations of the intestinal innate immune system and barrier functions may play a role in the pathogenesis of NEC itself.

Pic, an Autotransporter Protein Secreted by Different Pathogens in the Enterobacteriaceae Family, Is a Potent Mucus Secretagogue

A hallmark of enteroaggregative Escherichia coli (EAEC) infection is a formation of biofilm, which comprises a mucus layer with immersed bacteria in the intestines of patients. While studying the mucinolytic activity of Pic in an in vivo system, rat ileal loops, we surprisingly found that EAEC induced hypersecretion of mucus, which was accompanied by an increase in the number of mucus-containing goblet cells. Interestingly, an isogenic pic mutant (EAEC Δpic) was unable to cause this mucus hypersecretion. Furthermore, purified Pic was also able to induce intestinal mucus hypersecretion, and this effect was abolished when Pic was heat denatured. Site-directed mutagenesis of the serine protease catalytic residue of Pic showed that, unlike the mucinolytic activity, secretagogue activity did not depend on this catalytic serine protease motif. Other pathogens harboring the pic gene, such as Shigella flexneri and uropathogenic E. coli (UPEC), also showed results similar to those for EAEC, and construction of isogenic pic mutants of S. flexneri and UPEC confirmed this secretagogue activity. Thus, Pic mucinase is responsible for one of the pathophysiologic features of the diarrhea mediated by EAEC and the mucoid diarrhea induced by S. flexneri.

Interaction between bacterial peptides, neutrophils and goblet cells: a possible mechanism for neutrophil recruitment and goblet cell depletion in colitis

Goblet cell depletion occurs in various forms of colitis, but its mechanism is unknown. We have investigated two linked hypotheses: (i) that bacterial peptides, such as formyl-methionyl-leucyl-phenylalanine (fMLP), interact with epithelial cells inducing the release of chemokines, including interleukin-8 (IL-8), which in turn leads to the recruitment of neutrophils which release mucin secretagogues; (ii) that fMLP acts directly on epithelial cells to cause mucus secretion. Studies were performed to measure the effects of fMLP on the synthesis and secretion of IL-8 and mucus by the goblet cell differentiated colon cancer cell lines HT29-MTX (methotrexate-conditioned HT29 colonic adenocarcinoma cell line) and LS174T, and to assess the effects of neutrophil-derived secretagogues on goblet cell secretion in these cell lines. fMLP (0.1 microM) increased the secretion of IL-8 by 105% (P<0.0001) in HT29-MTX cells and by 401% (P<0.0001) in LS174T cells. fMLP also increased the synthesis and secretion of mucins by these cell lines, with maximal effects of 65% above control values for synthesis (P<0.01) and 73% for secretion (P<0.01). A dose-related increase (up to 67%; P<0.01) in mucin secretion was demonstrated in HT29-MTX cells in response to incubation with supernatant from activated neutrophils. This effect was largely (83%; P<0.02) inhibited by ICI 200,355, a specific inhibitor of neutrophil elastase. In conclusion, the bacterial peptide fMLP and neutrophil elastase are both potent mucus secretagogues for colon epithelial cells. fMLP also elicits release of the potent neutrophil chemoattractant IL-8 from colon epithelial cells. These findings support the hypothesis that the mucosal inflammation and mucus depletion seen in ulcerative colitis could result from interaction between bacterial peptides and the mucosa.

Regulation of secretion from mucous and serous cells in the excised ferret trachea

Mucus hypersecretion is an important characteristic of many airway diseases. Mucin is the major component of mucus, and is secreted from surface goblet cells of the airway epithelium and mucous cells of submucosal glands. Lysozyme is an enzyme secreted by serous cells of airway submucosal glands. We hypothesized that secretagogues acting through different pathways would have different effects on tracheal mucin and lysozyme secretion. We used a sandwich enzyme-linked lectin assay (ELLA) to measure mucin-like glycoprotein secretion and a spectrophotometric method to measure lysozyme secretion from isolated ferret tracheal segments. We evaluated the secretory response to four secretagogues; prostaglandin F 2α (PGF 2α), adenosine triphosphate (ATP), methacholine (MCh), and human neutrophil elastase (HNE). Each agent stimulated mucin and lysozyme secretion. The relative potency was PGF 2α≤ATP<MCh<HNE for mucin and ATP≤PGF 2α<MCh<HNE for lysozyme secretion. We showed that there is an anatomic gradient for constitutive and stimulated mucin and lysozyme secretion with the distal tracheal segments secreting more mucin and lysozyme per gram of tissue than the proximal segments. This robust model system can be used to evaluate the regulation of airway mucous and serous cell secretion and to assess the effect of agents that might alter the secretory response. We confirm that on an equimolar basis, HNE is one of the most potent mucus secretagogues.

Effect of bile salts on colonic mucus secretion in isolated vascularly perfused rat colon.

Mainly composed of mucins, mucus secreted by goblet cells in the intestinal epithelium is critically involved in the protection of the gastrointestinal mucosa. The hypothesis that bile and some bile salts can induce mucus secretion was tested in the isolated perfused rat colon. Mucus release was evaluated using enzyme-linked immunosorbent assays and supported by histological analysis. Luminal administration of bile extract (1%) provoked mucus secretion in the rat colon. Deoxycholate (0.5-10 mM) induced a dose-dependent increase in rat colonic mucus release. Chenodeoxycholate (10 mM) and hyodeoxycholate (10 mM) also evoked mucus discharge, whereas 10 mM cholate, 10 mM ursodeoxycholate, or Tween-20 did not release mucus. Taurine-conjugated bile salts (deoxycholate, hyodeoxycholate, and chenodeoxycholate) were less potent mucus secretagogues than the corresponding unconjugated forms. The deoxycholate-induced mucus discharge was not altered by pharmacological blockers (tetrodotoxin, atropine), indomethacin, mast cell stabilizers (ketotifen, doxantrazole), H1 histamine receptor antagonist (pyrilamine), or 5-HT receptor antagonists (ketanserin, ondansetron, SDZ 205-557). Our findings suggest that some bile salts, especially in the unconjugated form, may provoke colonic mucus secretion, probably through a direct action on mucus-secreting cells.

Mucin discharge from the isolated vascularity perfused rat colon in response to bile salts

The intestinal mucus layer plays an important role in the protection of the mucosa. This function is performed mainly by mucins, which are high molecular-weight glycoproteins produced by goblet cells. Luminal factors that govern mucin secretion are poorly known. The hypothesis that bile salts could induce colonic mucin discharge was thus tested in a model of isolated vascularly perfused rat colon. Methods : Ligated loops (I O-cm length) of rat colon were separated from adjacent tissue, transferred to a saline bath and perfused through the superior mesenteric artery with a Krebs Henseleit buffer containing 25% washed bovine erythrocytes, 3% bovine albumin, amino acids and glucose. After a lO-min control period, loops were filled with Iml of prewarmed saline containing stimulants. After 30-min period of stimulation, mucus was assayed in luminal content by enzyme-linked immunosorbent assay. Tissue samples from perfused rat colon were also immersed in Karnovsky's fixative for subsequent staining of mucus cells with alcian blue followed by the periodic acid schiff reagent. Results : Luminal administration of deoxycholate (I-lOmM) produced a dose-dependent release of mucins (maximal response at 550 % of control loops) in the isolated perfused rat colon. Hyodeoxycholate (IOmM) and Chenodeoxycholate (I0mM) also induced an increase in mucin discharge (response at 310 % and 290 % of controls,respectively) whereas IOmM cholate, ursodeoxycholate and tween-20 were without effect. Taurine conjugated bile salts (deoxycholate,hyodeoxycholate, and chenodeoxycholate) were less potent mucus secretagogues than unconjugated forms. Tetrodotoxin, atropine, indomethacin, a mast cells stabilizer (ketotifen), H I histamine antagonist(pyrilamine) and 5-HT receptors antagonists (ketanserin, SDZ, Zophren) did not significantly modify deoxycholate-induced mucin secretion. Conclusions : The present study conducted with the isolated vascularly perfused rat colon suggests that some bile salts may playa role in the regulation of colonic mucin secretion by way of a direct action on mucus cells

Human neutrophil elastase releases two pools of mucinlike glycoconjugate from tracheal submucosal gland cells.

Neutrophil elastase can contribute to the pathogenesis of increased airway reactivity and excess mucus secretion in many pulmonary diseases. Ten nanomolar human neutrophil elastase (HNE) effectively empties airway serous cells, raising the question of why HNE is not equally effective at emptying mucous cells of their stored mucin because total release of mucin granules is not seen in postmortem examination of even the most severe disease. To better resolve the mucus secretagogue action of HNE, we measured secretion of mucinlike glycoconjugates (MGCs) released from freshly isolated swine tracheal submucosal gland cells in fractions of the superfusate acquired every 2 min. Six to fifty nanomolar HNE released a fixed quantity of MGCs at an increasing rate with increasing concentrations of enzyme, an action consistent with the release of cell surface mucinlike molecules. The polycation poly-L-lysine (1 microg/ml) released a similar transient of MGCs. A steady-state doubling of MGC rate of release was seen as long as 100 nM HNE was present, but this stimulus represented less than a 1% release of stored MGCs/min and was consistent with release of mucin vesicles from cell stores. Both actions of HNE were inhibited by the specific inhibitors L-680833 and DMP-777 but not by 30 microM erythromycin. Therefore, HNE release of MGCs from tracheal submucosal glands is limited by both the fixed quantity of the MGCs in the transient pool and by the small steady-state response to the higher concentrations of enzyme.

Regulation of a mucus secretagogue by somatostatin in a human macrophage hybridoma cell line

Regulation of a mucus secretagogue by somatostatin in a human macrophage hybridoma cell line

Effect of anion transport inhibition on mucus secretion by airway submucosal glands.

To model the airway glandular defect in cystic fibrosis (CF), the effect of anion secretion blockers on submucosal gland mucus secretion was investigated. Porcine distal bronchi were isolated, pretreated with a Cl- secretion blocker (bumetanide) and/or a combination of blockers to inhibit HCO3- secretion (dimethylamiloride, acetazolamide, and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), and then treated with acetylcholine (ACh), a glandular liquid and mucus secretagogue. Bronchi were then fixed, sectioned, and stained for mucins. Each gland duct was ranked for mucin content from zero (no mucin) to five (duct completely occluded with mucin). Untreated bronchi, bronchi treated only with ACh, and ACh-treated bronchi that received either bumetanide or the HCO3- secretion blockers all exhibited low gland duct mucin content (1.18 +/- 0.34, 0.59 +/- 0.07, 0.65 +/- 0.03, and 0.83 +/- 0.11, respectively). However, pretreatment with both Cl- and HCO3- secretion blockers before ACh addition resulted in substantial and significant ductal mucus accumulation (3.57 +/- 0.22). In situ videomicroscopy studies of intact airways confirmed these results. Thus inhibition of the anion (and presumably liquid) secretion response to ACh leads to mucus obstruction of submucosal gland ducts that resembles the early pathological changes observed in CF.

Detection of a novel macrophage-derived mucus secretagogue (MMS-68) in bronchoalveolar lavage fluid of patients with asthma

Background: We have previously described a novel high-molecular-weight macrophage-derived mucus secretagogue (MMS-68) that causes mucuslike glycoconjugate release from cultured airways, nasal explants, and the Ishikawa adenocarcinoma cell line. We have generated monoclonal antibodies against MMS-68 and have developed an antigen-capture ELISA to measure MMS-68 levels in biologic fluids. Using this ELISA, we have demonstrated elevated levels of MMS-68 in the bronchoalveolar lavage fluid (BALF) of smokers and persons chronic bronchitis, in a patient with asthma and bronchorrhea, and in nasal lavage from patients with allergic rhinitis challenged with histamine and methacholine. We have also demonstrated that both spontaneous and lipopolysaccharide induced MMS-68 production is increased in the culture supernatants of monocytes from patients with steroid-dependent asthmas compared with those from normal control subjects. Methods: To delineate further a role for MMS-68 in the regulation of mucus secretion in asthma, we measured MMS-68 levels in the BALF of 37 patients with non-steroid-dependent asthma and of 16 control subjects. Results: There were 21 men and 16 women in the asthma group (age range, 17 to 62 years; mean, 33.8 years) and 11 men and five women in the control group (age range, 18 to 42 years; mean, 27.8 years). There were no statistical differences in either total cell count (145.5 × 103 ± 75.7 cells/mm 3 × 10 3 cells/mm 3 vs 134 × 103 ± 65.9 × 10 3 cells/mm 3, p < 0.234) or numbers of alveolar macrophages (103.7 × 10 3 ± 71.7 × 10 3/mm 3 vs 98.7 × 10 3 ± 65 × 10 3 cells/mm 3, p < 0.244) when the asthmatic group was compared with the control group. The MMS-68 level in the asthmatic group was 2.1 ± 0.25 μg MMS-68 per milligram protein compared with 2.09 ± 0.26 μg MMS-68 per milligram protein ( p < 0.256) in the control group. Conclusions: There was no correlation between MMS-68 levels and total protein content, numbers of alveolar macrophages, or the production of other macrophage-derived cytokines including interleukin-1, interleukin-6, or tumor necrosis factor in the asthmatic BALF. Mild asthma, which is clinically not associated with mucus hypersecretion, was not associated with elevated levels of MMS-68. We believe that direct correlation exists between mucus hypersecretion and MMS-68 levels. (J A LLERGY C LIN I MMUNOL 1995;95:868-76)

Characterization of a unique mucin-like glycoprotein secreted by a human endometrial adenocarcinoma cell line (Ishikawa).

A human endometrial adenocarcinoma cell line (Ishikawa) has been shown to incorporate [3H]glucosamine and to secrete a radiolabeled high molecular weight compound which is excluded from a Sepharose CL-2B column. The excluded material was resistant to hyaluronidase, chondroitinase ABC, and heparinase. These findings rule out the possibility of this material being a proteoglycan. The susceptibility of this material to digestion with pronase, neuraminidase, and alkaline borohydride treatment strongly suggests that the excluded material is an O-glycosidic glycoprotein. The glycoprotein secreted by Ishikawa cells (ICGP) did not react immunologically with antibodies against either lactoferrin or fibronectin, but did react with an antibody made against tracheal mucin. Conversely, immunoblot analysis revealed that an antibody made against ICGP did not recognize hyaluronic acid, chondroitin, heparin, nasal turbinate mucin, bovine submaxillary gland mucin, lactoferrin, or fibronectin, but did recognize tracheal mucin. Analysis of ICGP amino acid and carbohydrate composition showed that it is rich in serine, threonine, glutamic acid, aspartic acid, and N-acetylneuraminic acid. In this respect, ICGP differs from other mucins, even though it is immunologically similar to respiratory mucin; hence we may consider ICGP to be a mucin-like glycoprotein. Secretion of ICGP can be modulated by Ca(2+)-ionophore and other mucus secretagogues, such as platelet activating factor, carbachol, and monocyte/macrophage mucus secretagogue, all mediators of lung inflammation. Ishikawa cells and anti-ICGP antibody may be used in studies on in vitro regulation of mucin-like glycoprotein synthesis and secretion in the respiratory tract as well as in the endometrium.

The gastroduodenal mucus barrier and its role in protection against luminal pepsins: the effect of 16,16 dimethyl prostaglandin E2, carbopol-polyacrylate, sucralfate and bismuth subsalicylate.

Mucus and bicarbonate secretions have been widely implicated as an important pre-epithelial protective barrier against autodigestion of the gastric mucosa by acid and pepsin. Evidence from several independent studies shows there is a continuous layer of resilient viscoelastic mucus gel adherent to the surface of the gastroduodenal mucosa. The median thickness of the adherent gastric mucus layer in humans is 180 microns, range 50-450 microns. The epithelial bicarbonate secretion permeates the unstirred matrix of mucus gel neutralizing luminal acid and establishing a pH gradient within the gel. In the duodenum, evidence supports the mucus bicarbonate barrier as a major protective mechanism against acid aggression. The adherent mucus gel, by acting as an effective 'permeability' barrier to pepsin, protects the underlying sensitive mucosa from digestion. However, pepsin slowly digests mucus gel at its luminal surface to produce soluble degraded mucin. In a rat gastric damage model in vivo, pepsin in excess digests the gastric mucus barrier sufficiently rapidly to outweigh new mucus secretion and lead to breaching of the mucus barrier with the formation of small punctate ulcers in the epithelium accompanied by mucosal haemorrhage. The mucus secretagogue 16,16 dimethyl prostaglandin E2 and the muco-adhesive carbopol-polyacrylate both fully protected the mucosa against pepsin damage by enhancing the protective properties of the mucus barrier. Sucralfate and bismuth subsalicylate were partially effective in protection against pepsin damage but this protection was mainly mediated at the level of the mucosa. In peptic ulcer disease, there is increased mucolytic (mucus degrading) activity in gastric juice and this is associated with an impaired mucin polymeric structure and a weaker mucus barrier.(ABSTRACT TRUNCATED AT 250 WORDS)

In vivo detection of a novel macrophage-derived protein involved in the regulation of nasal mucus-like glycoconjugate secretion

Background: We recently described a novel 68 kd mucus secretagogue (MMS-68) derived from human monocytes, pulmonary macrophages, and a macrophage hybridoma, clone 63. We detected MMS-68 in monocyte culture supernatants from patients with steroid-dependent asthma and in bronchoalveolar lavage fluid from patients with chronic bronchitis by antigen capture ELISA and in normal lung tissue by immunohistochemistry. Methods: To determine a role for MMS-68 in the regulation of nasal mucus, we labeled human nasal turbinates with tritiated glucosamine and assayed for the ability of the previously purified MMS-68 (stock solution) to induce mucus-like glycoconjugate release (MLGC). We also performed immunohistochemistry stains with an anti-MMS-68 antibody (1-D-10) on frozen sections ( n = 5) of nasal turbinates from patients with allergic and nonallergic rhinitis who were undergoing rhinoplasty and measured MMS-68 levels in nasal lavages from patients who were undergoing topical nasal histamine or methacholine challenge. Results: MMS-68 is a potent nasal MLGC secretagogue causing a dose-dependent increase in MLGC release in vitro. Staining revealed a subepithelial distribution for MMS-68. Antigen capture ELISA of nasal lavages demonstrated mean MMS-68 levels from saline control challenge of 0.9 ± 0.5 μg MMS-68 per milligram of protein ( n = 5), 8.6 ± 1.4 μg MMS-68 per milligram of protein from histamine challenge and 20.7 ± 2.3 μg MMS-68 per milligram of protein ( n = 5) after methacholine challenge. Conclusion: Taken together these data suggest that MMS-68 may play a role in the normal regulation of mucus secretion.