Effects of matrix metalloproteinase inhibitor on LPS-induced goblet cell metaplasia

Bacterial infections of the lung are known to induce inflammatory responses, which lead to mucus hypersecretion. Moreover, mucin synthesis in the airways has been reported to be regulated by neutrophilic inflammation-induced epidermal growth factor receptor (EGFR) expression and its activation. Furthermore, matrix metalloproteinases (MMPs), especially MMP-9, have been reported to promote the transmigration of activated neutrophils. In this study, we investigated the associations between lipopolysaccharide (LPS)-induced goblet cell (GC) metaplasia and EGFR expression and the effects of MMP inhibitor (MMPI). Various concentrations of LPS were instilled into the tracheas of pathogen-free Sprague-Dawley rats, and airways were examined at different times after LPS instillation. To examine the role of MMP-9, we treated rats 3 days before LPS instillation and daily thereafter with MMPI. Neutrophilic infiltration, Alcian blue/periodic acid-Schiff (AB/PAS) staining, and immunohistochemical staining for MUC5AC, EGFR, and MMP-9 were performed. The instillation of LPS increased AB/PAS and MUC5AC staining in time- and dose-dependent manners, and treatment with MMPI significantly prevented GC metaplasia. The instillation of LPS into the trachea also induced neutrophilic infiltration and EGFR and MMP-9 expression in the airway epithelium, and MMPI was found to significantly prevent neutrophil recruitment, GC metaplasia, and EGFR and MMP-9 expression. This study demonstrates that the MMP-9 and EGFR cascades are associated with LPS-induced mucus hypersecretion.     

PAF mediates cigarette smoke-induced goblet cell metaplasia in guinea pig airways

Goblet cell metaplasia is an important morphological feature in the airways of patients with chronic airway diseases; however, the precise mechanisms that cause this feature are unknown. We investigated the role of endogenous platelet-activating factor (PAF) in airway goblet cell metaplasia induced by cigarette smoke in vivo. Guinea pigs were exposed repeatedly to cigarette smoke for 14 consecutive days. The number of goblet cells in each trachea was determined with Alcian blue-periodic acid-Schiff staining. Differential cell counts and PAF levels in bronchoalveolar lavage fluid were also evaluated. Cigarette smoke exposure significantly increased the number of goblet cells. Eosinophils, neutrophils, and PAF levels in bronchoalveolar lavage fluid were also significantly increased after cigarette smoke. Treatment with a specific PAF receptor antagonist, E-6123, significantly attenuated the increases in the number of airway goblet cells, eosinophils, and neutrophils observed after cigarette smoke exposure. These results suggest that endogenous PAF may play a key role in goblet cell metaplasia induced by cigarette smoke and that potential roles exist for inhibitors of PAF receptor in the treatment of hypersecretory airway diseases.     

Neonatal rhinovirus infection induces mucous metaplasia and airways hyperresponsiveness

Recent studies link early rhinovirus (RV) infections to later asthma development. We hypothesized that neonatal RV infection leads to an IL-13-driven asthma-like phenotype in mice. BALB/c mice were inoculated with RV1B or sham on day 7 of life. Viral RNA persisted in the neonatal lung up to 7 d postinfection. Within this time frame, IFN-α, -β, and -γ peaked 1 d postinfection, whereas IFN-λ levels persisted. Next, we examined mice on day 35 of life, 28 d after initial infection. Compared with sham-treated controls, virus-inoculated mice demonstrated airways hyperresponsiveness. Lungs from RV-infected mice showed increases in several immune cell populations, as well as the percentages of CD4-positive T cells expressing IFN-γ and of NKp46/CD335(+), TCR-β(+) cells expressing IL-13. Periodic acid-Schiff and immunohistochemical staining revealed mucous cell metaplasia and muc5AC expression in RV1B- but not sham-inoculated lungs. Mucous metaplasia was accompanied by induction of gob-5, MUC5AC, MUC5B, and IL-13 mRNA. By comparison, adult mice infected with RV1B showed no change in IL-13 expression, mucus production, or airways responsiveness 28 d postinfection. Intraperitoneal administration of anti-IL-13 neutralizing Ab attenuated RV-induced mucous metaplasia and methacholine responses, and IL-4R null mice failed to show RV-induced mucous metaplasia. Finally, neonatal RV increased the inflammatory response to subsequent allergic sensitization and challenge. We conclude that neonatal RV1B infection leads to persistent airways inflammation, mucous metaplasia, and hyperresponsiveness, which are mediated, at least in part, by IL-13.     

Activation of epidermal growth factor receptors is responsible for mucin synthesis induced by cigarette smoke

Mucus hypersecretion from hyperplastic airway goblet cells is a hallmark of chronic obstructive pulmonary disease (COPD). Although cigarette smoking is thought to be involved in mucus hypersecretion in COPD, the mechanism by which cigarette smoke induces mucus overproduction is unknown. Here we show that activation of epidermal growth factor receptors (EGFR) is responsible for mucin production after inhalation of cigarette smoke in airways in vitro and in vivo. In the airway epithelial cell line NCI-H292, exposure to cigarette smoke upregulated the EGFR mRNA expression and induced activation of EGFR-specific tyrosine phosphorylation, resulting in upregulation of MUC5AC mRNA and protein production, effects that were inhibited completely by selective EGFR tyrosine kinase inhibitors (BIBX1522, AG-1478) and that were decreased by antioxidants. In vivo, cigarette smoke inhalation increased MUC5AC mRNA and goblet cell production in rat airways, effects that were prevented by pretreatment with BIBX1522. These effects may explain the goblet cell hyperplasia that occurs in COPD and may provide a novel strategy for therapy in airway hypersecretory diseases.     

IL-13-induced Clara cell secretory protein expression in airway epithelium: role of EGFR signaling pathway

Previous work showed that the Th2 cytokine interleukin (IL)-13 induces goblet cell metaplasia via an indirect mechanism involving the expression and subsequent activation of epidermal growth factor receptor (EGFR). Because Clara cell secretory protein (CCSP) expression has been reported in cells that express mucins, we examined the effect of IL-13 on CCSP gene and protein expression in pathogen-free rat airways and in pulmonary mucoepidermoid NCI-H292 cells. Intratracheal instillation of IL-13 induced CCSP mRNA in epithelial cells without cilia within 8-16 h, maximal between 24 and 48 h; CCSP immunostaining increased in a time-dependent fashion, maximal at 48 h. The CCSP immunostaining was localized in nongranulated secretory cells and goblet cells and in the lumen. Pretreatment with the selective EGFR tyrosine kinase inhibitor BIBX1522, cyclophosphamide (an inhibitor of bone marrow leukocyte mobilization), or a blocking antibody to IL-8 prevented CCSP staining. Treatment of NCI-H292 cells with the EGFR ligand transforming growth factor-alpha, but not with IL-13 alone, induced CCSP gene and protein expression. Selective EGFR tyrosine kinase inhibitors, BIBX1522 and AG1478, prevented CCSP expression in NCI-H292 cells, but the platelet-derived growth factor receptor tyrosine kinase inhibitor AG1295 had no effect. These findings indicate that IL-13 induces CCSP expression via an EGFR- and leukocyte-dependent pathway.     

Effect of MMP/ADAM inhibitors on goblet cell hyperplasia in cultured human bronchial epithelial cells

While epidermal growth factor receptor (EGFR) plays a pivotal role in the repair process of epithelial cells, it is also involved in the overproduction of mucus and goblet cell hyperplasia (GCH), which occurs in chronic airway diseases such as asthma. Among the EGFR ligands, transforming growth factor (TGF)-alpha is thought to be the most important in the synthesis of mucus. Pro-TGF-alpha is cleaved to give an active form by members of the matrix metalloproteinases (MMP)/a disintegrin and metalloproteinases (ADAM) family. Thus MMP/ADAM inhibitors might prevent GCH by inhibiting transactivation of EGFR. Upon stimulation of differentiating normal human bronchial epithelial (NHBE) cells by IL-13, GCH was induced. The mucin genes MUC5AC, MUC5B, and MUC2 were upregulated whereas the expression of ciliated cell markers was greatly repressed. GM6001, a broad-spectrum inhibitor for MMP/ADAM, inhibited IL-13-induced mucin gene expression and mucus production as measured by periodic acid-Schiff staining. This was accompanied by an inhibition of TGF-alpha release. These results suggest that MMP/ADAMs play a pivotal role in the development of GCH in lung epithelial cells.     

Human eosinophils induce mucin production in airway epithelial cells via epidermal growth factor receptor activation.

Eosinophil recruitment and mucus hypersecretion are characteristic of asthmatic airway inflammation, but eosinophils have not been shown to induce mucin production. Because an epidermal growth factor receptor (EGFR) cascade induces MUC5AC mucin in airways, and because EGFR is up-regulated in asthmatic airways, we examined the effect of eosinophils on MUC5AC mucin production in NCI-H292 cells (a human airway epithelial cell line that produces mucins). Eosinophils were isolated from the peripheral blood of allergic patients, and their effects on MUC5AC mucin gene and protein synthesis were assessed using in situ hybridization and ELISAs. When IL-3 plus GM-CSF or IL-3 plus IL-5 were added to eosinophils cultured with NCI-H292 cells, MUC5AC mucin production increased; eosinophils or cytokines alone had no effect. Eosinophil supernatant obtained by culturing eosinophils with IL-3 plus GM-CSF or IL-3 plus IL-5 also increased MUC5AC synthesis in NCI-H292 cells, an effect that was prevented by selective EGFR inhibitors (AG1478, BIBX1522). Supernatant of activated eosinophils induced EGFR phosphorylation in NCI-H292 cells. Supernatant of activated eosinophils contained increased concentrations of TGF-alpha protein (an EGFR ligand) and induced up-regulation of TGF-alpha expression and release in NCI-H292 cells. A blocking Ab to TGF-alpha reduced activated eosinophil-induced MUC5AC synthesis in NCI-H292 cells. These results show that activated eosinophils induce mucin synthesis in human airway epithelial cells via EGFR activation, and they implicate TGF-alpha produced by eosinophils and epithelial cells in the EGFR activation that results in mucin production in human airway epithelium.     

IL-13 induces mucin production by stimulating epidermal growth factor receptors and by activating neutrophils

Mucus hypersecretion contributes to the morbidity and mortality in acute asthma. Both T helper 2 (Th2) cytokines and epidermal growth factor receptor (EGFR) signaling have been implicated in allergen-induced goblet cell (GC) metaplasia. Present results show that a cascade of EGFR involving neutrophils is implicated in interleukin (IL)-13-induced mucin expression in GC. Treatment with a selective EGFR tyrosine kinase inhibitor prevented IL-13-induced GC metaplasia dose dependently and completely. Instillation of IL-13 also induced tumor necrosis factor-alpha protein expression, mainly in infiltrating neutrophils. Control airway epithelium contained few leukocytes, but intratracheal instillation of IL-13 resulted in time-dependent leukocyte recruitment by IL-13-induced IL-8-like chemoattractant expression in airway epithelium. Pretreatment with an inhibitor of leukocytes in the bone marrow (cyclophosphamide) or with a blocking antibody to IL-8 prevented both IL-13-induced leukocyte recruitment and GC metaplasia. These findings indicate that EGFR signaling is involved in IL-13-induced mucin production. They suggest a potential therapeutic role for inhibitors of the EGFR cascade in the hypersecretion that occurs in acute asthma.     

Distinct roles for the NK cell-activating receptors in mediating interactions with dendritic cells and tumor cells

Hyperproduction of goblet cells and mucin in the airway epithelium is an important feature of airway inflammatory diseases. We investigated the involvement of Notch signaling in MUC5AC expression in NCI-H292 cells, a human lung carcinoma cell line. Epidermal growth factor (EGF) stimulated generation of the Notch intracellular domain (NICD) in a RBP-Jκ-dependent manner. Treatment with γ-secretase inhibitors L-685,458 or DAPT or introduction of small interfering RNA directed against Notch1 reduced EGF-induced MUC5AC expression. The inhibitory effect of L-685,458 on EGF-induced MUC5AC mRNA and protein expression was also observed in primary human bronchial epithelial cells. Blockage of Notch signaling with L-685,458 or Notch siRNA resulted in a decrease in EGF-induced phosphorylation of ERK. These results suggested that ERK activation is necessary for the regulation of EGF receptor (EGFR)-mediated MUC5AC expression by Notch signaling. Conversely, forced expression of NICD induced both EGFR and ERK phosphorylation with MUC5AC expression even in the absence of EGF. Treatment of the NICD-expressing cells with EGF further augmented ERK phosphorylation in an additive manner. The ERK phosphorylation induced by exogenous NICD was inhibited by treatment with an Ab that antagonizes EGFR activity as well as by inhibitors of EGFR and ERK, implying that Notch signaling induces MUC5AC expression by activating the EGFR pathway. Collectively, these results suggest that MUC5AC expression is regulated by a bidirectional circuit between Notch and EGFR signaling pathways.     

IL-4 induces mucin gene expression and goblet cell metaplasia in vitro and in vivo

Goblet cell metaplasia and mucus hypersecretion are important features in the pathogenesis of asthma. The cytokine IL-4 has been shown to play a role in animal models of asthma, where it induces Th2 lymphocyte differentiation and B lymphocyte IgE class switch. IL-4 has also been implicated in the differentiation of goblet cells via effects on lymphocytes and eosinophils. In this study we hypothesized that IL-4 induces airway epithelial cell mucin gene expression and mucous glycoconjugate production by direct action on these cells. In vitro, cultured airway epithelial cells (NCI-H292) expressed IL-4R constitutively, and IL-4 (10 ng/ml) induced MUC2 gene expression and mucous glycoconjugate production. In vivo, mouse airway epithelial cells expressed IL-4R constitutively, and IL-4 (250 ng) increased MUC5 gene expression and Alcian blue/periodic acid-Schiff-positive staining at 24 h; IL-4 did not increase inflammatory cell numbers in airway tissue or in bronchoalveolar lavage. TNF-alpha and IL-1beta levels in bronchoalveolar lavage were not increased in response to IL-4 instillation. These results indicate that airway epithelial cells express IL-4R constitutively and that IL-4 directly induces the differentiation of epithelium into mucous glycoconjugate-containing goblet cells.     

Effect of MMP/ADAM Inhibitors on Goblet Cell Hyperplasia in Cultured Human Bronchial Epithelial Cells

While epidermal growth factor receptor (EGFR) plays a pivotal role in the repair process of epithelial cells, it is also involved in the overproduction of mucus and goblet cell hyperplasia (GCH), which occurs in chronic airway diseases such as asthma. Among the EGFR ligands, transforming growth factor (TGF)-α is thought to be the most important in the synthesis of mucus. Pro-TGF-α is cleaved to give an active form by members of the matrix metalloproteinases (MMP)/a disintegrin and metalloproteinases (ADAM) family. Thus MMP/ADAM inhibitors might prevent GCH by inhibiting transactivation of EGFR. Upon stimulation of differentiating normal human bronchial epithelial (NHBE) cells by IL-13, GCH was induced. The mucin genes MUC5AC, MUC5B, and MUC2 were upregulated whereas the expression of ciliated cell markers was greatly repressed. GM6001, a broad-spectrum inhibitor for MMP/ADAM, inhibited IL-13-induced mucin gene expression and mucus production as measured by periodic acid-Schiff staining. This was accompanied by an inhibition of TGF-α release. These results suggest that MMP/ADAMs play a pivotal role in the development of GCH in lung epithelial cells.     

Role of epidermal growth factor receptor activation in regulating mucin synthesis

Healthy individuals have few goblet cells in their airways, but in patients with hypersecretory diseases goblet-cell upregulation results in mucus hypersecretion, airway plugging, and death. Multiple stimuli produce hypersecretion via epidermal growth factor receptor (EGFR) expression and activation, causing goblet-cell metaplasia from Clara cells by a process of cell differentiation. These cells are also believed to be the cells of origin of non-small-cell lung cancer, but this occurs via cell multiplication. The mechanisms that determine which pathway is chosen are critical but largely unknown. Although no effective therapy exists for hypersecretion at present, the EGFR cascade suggests methods for effective therapeutic intervention.     

ATP induced MUC5AC release from human airways in vitro

BACKGROUND: Chronic airway diseases are often associated with marked mucus production, however, little is known about the regulation of secretory activity by locally released endogenous mediators. AIM: This investigation was performed to determine the release of MUC5AC mucin from human bronchial preparations using the purinergic agonists adenosine 5''-triphosphate (ATP) and uridine 5''-triphosphate (UTP). METHODS: Immunohistochemical and immunoradiometric assays (IRMA) were used to detect the MUC5AC mucin. Immunohistochemical analysis were performed using individual 1-13 M1 and 21 M1 MAbs recognizing a recombinant M1 mucin partially encoded by the MUC5AC gene. IRMA measurments were performed using a mixture of eight anti-M1 mucin MAbs (PM8), which included both 1-13 M1 and 21 M1 MAbs. Lysozyme and protein were also measured in the biological fluids derived from human bronchial preparations obtained from patients who had undergone surgery for lung carcinoma. RESULTS: The anti-M1 monoclonal antibodies labelled epithelial goblet cells. After challenge of human bronchial preparations with ATP, the goblet cells exhibited less staining. In contrast, UTP did not alter the immunolabelling of goblet cells. MUC5AC mucin in the bronchial fluids derived from ATP-challenged preparations was increased while UTP had no effect on release. ATP did not alter either the quantities of lysozyme or protein detected in the biological fluids. CONCLUSION: These results suggest that ATP may regulate epithelial goblet cell secretion of MUC5AC mucin from human airways in vitro.     

Regulation of PMA-induced MUC5AC expression by heparin in human bronchial epithelial cells

Mucus hypersecretion is a major pathophysiologic feature in chronic inflammatory airway diseases. Oxidative stress plays a pivotal role in this process. Recent studies have found that heparin has antioxidant effects which can reduce free radical damage. Here, we hypothesized that heparin has some influence on the expression of mucin 5AC (MUC5AC) induced by phorbol myristate acetate (PMA) in a bronchial epithelial cell line (HBE16), also we have investigated the potential mechanism involved in the process. We found that ROS, the mRNA of Duox1, EGFR and MUC5AC, as well as the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC in the PMA group were significantly increased when compared with the control group (all P < 0.01). After pretreatment with heparin however, there was a significant decrease in ROS levels, the mRNA of Duox1, EGFR, and MUC5AC, and the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC, when compared with the PMA group (all P < 0.01). MUC5AC protein in the supernatant was inhibited in a dose-dependent manner by heparin. Pretreatment with DMTU resulted in a significant decrease in ROS content, the mRNA of Duox1, EGFR, and MUC5AC as well as the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC when compared with the PMA group (all P < 0.01). When cells were pretreated with both heparin and DMTU, there was a further reduction in ROS content, the mRNA of Duox1, EGFR, and MUC5AC as well as the protein levels of Duox1, p-EGFR, EGFR, and MUC5AC, when compared with either the PMA group, heparin group, or DMTU group (all P < 0.01). Our results show that PMA can induce MUC5AC expression by activation of the Duox1-ROS-TACE-TGF-α-EGFR signaling pathway. Heparin can decrease the level of Duox1, ROS production and block the PMA-induced activation of EGFR, thus inhibiting the overexpression of mucin MUC5AC in a dose-dependent manner. In addition to reducing ROS production, heparin may also inhibit the expression of MUC5AC through other signal mechanisms.     

The Idiopathic Pulmonary Fibrosis Honeycomb Cyst Contains A Mucocilary Pseudostratified Epithelium

Background

We previously identified a MUC5B gene promoter-variant that is a risk allele for sporadic and familial Idiopathic Pulmonary Fibrosis/Usual Interstitial Pneumonia (IPF/UIP). This allele was strongly associated with increased MUC5B gene expression in lung tissue from unaffected subjects. Despite the strong association of this airway epithelial marker with disease, little is known of mucin expressing structures or of airway involvement in IPF/UIP.

Methods

Immunofluorescence was used to subtype mucus cells according to MUC5B and MUC5AC expression and to identify ciliated, basal, and alveolar type II (ATII) cells in tissue sections from control and IPF/UIP subjects. Staining patterns were quantified for distal airways (Control and IPF/UIP) and in honeycomb cysts (HC).

Results

MUC5B-expressing cells (EC) were detected in the majority of control distal airways. MUC5AC-EC were identified in half of these airways and only in airways that contained MUC5B-EC. The frequency of MUC5B+ and MUC5AC+ distal airways was increased in IPF/UIP subjects. MUC5B-EC were the dominant mucus cell type in the HC epithelium. The distal airway epithelium from control and IPF/UIP subjects and HC was populated by basal and ciliated cells. Most honeycombing regions were distinct from ATII hyperplasic regions. ATII cells were undetectable in the overwhelming majority of HC.

Conclusions

The distal airway contains a pseudostratified mucocilary epithelium that is defined by basal epithelial cells and mucus cells that express MUC5B predominantly. These data suggest that the HC is derived from the distal airway.     

E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC mucin expression in cultured human airway epithelial cells

In previous work, we showed that epidermal growth factor receptor (EGFR) activation causes mucin expression in airway epithelium in vivo and in human NCI-H292 airway epithelial cells and normal human bronchial epithelial (NHBE) cells in vitro. Here we show that the cell surface adhesion molecule, E-cadherin, promotes EGFR-mediated mucin production in NCI-H292 cells in a cell density- and cell cycle-dependent fashion. The addition of the EGFR ligand, transforming growth factor (TGF)-alpha, increased MUC5AC protein expression markedly in dense, but not in sparse, cultures. MUC5AC-positive cells in dense cultures contained 2 N DNA content and did not incorporate bromodeoxyuridine, suggesting that they develop via cell differentiation and that a surface molecule involved in cell-cell contact is important for EGFR-mediated mucin production. In support of this hypothesis, in dense cultures of NCI-H292 cells and in NHBE cells at air-liquid interface, blockade of E-cadherin-mediated cell-cell contacts decreased EGFR-dependent mucin production. E-cadherin blockade also increased EGFR-dependent cell proliferation and TGF-alpha-induced EGFR tyrosine phosphorylation in dense cultures of NCI-H292 cells, suggesting that E-cadherin promotes EGFR-dependent mucin production and inhibits EGFR-dependent cell proliferation via modulation of EGFR phosphotyrosine levels. Furthermore, in dense cultures, E-cadherin blockade decreased the rate of EGFR tyrosine dephosphorylation, implicating an E-cadherin-dependent protein tyrosine phosphatase in EGFR dephosphorylation. Thus E-cadherin promotes EGFR-mediated cell differentiation and MUC5AC production, and our results suggest that this occurs via a pathway involving protein tyrosine phosphatase-dependent EGFR dephosphorylation.     

Neutrophil elastase induces MUC5AC mucin production in human airway epithelial cells via a cascade involving protein kinase C, reactive oxygen species, and TNF-alpha-converting enzyme

Mucus hypersecretion is a prominent manifestation in patients with chronic inflammatory airway diseases and contributes to their morbidity and mortality by plugging airways and causing recurrent infections. Human neutrophil elastase (HNE) exists in high concentrations (1-20 microM) in airway secretions of these patients and induces overproduction of MUC5AC mucin, a major component of airway mucus. Previous studies showed that HNE induces MUC5AC mucin production involving reactive oxygen species (ROS) generation and TGF-alpha-dependent epidermal growth factor receptor (EGFR) activation in human airway epithelial cells. However, the molecular mechanisms involved in these responses are not defined. TNF-alpha-converting enzyme (TACE) cleaves pro-TGF-alpha into soluble TGF-alpha and can be activated by ROS. We hypothesize that HNE activates TACE via ROS generation, resulting in cleavage of pro-TGF-alpha, EGFR activation, and MUC5AC mucin expression in airway epithelial cells. Here we show that in human airway epithelial cells HNE increases TGF-alpha release, EGFR phosphorylation, and MUC5AC mucin expression, effects that were attenuated by TACE inhibitor TAPI-1 and by specific knockdown of TACE expression with small interfering RNA, implicating TACE in HNE-induced responses. These responses to HNE were also reduced by pretreatment with ROS scavengers, implicating ROS. Furthermore, we show that HNE causes protein kinase C (PKC) activation and translocation from cytosol to plasma membrane; blockade of this effect by PKC inhibitors reduced HNE-induced ROS generation and other responses, implicating PKC. We conclude that HNE induces MUC5AC mucin expression via a cascade involving PKC-ROS-TACE in human airway epithelial cells.     

Identification of glycoproteins in goblet cells of epidermis and gill of plaice (Pleuronectes platessa L.), flounder (Platichthys flesus (L.)) and rainbow trout (Salmo gairdneri Richardson)

Synopsis A quantitative analysis has been made of the glycoproteins present in the goblet cells of the epidermis, gill filaments and gill lamellae of three species of teleost fish. The glycoproteins have been identified by a combination of techniques, including the use of the enzyme sialidase followed by Alcian Blue staining, at pH 2.6 or I. o, in combination with periodic acid-Schiff. The selected fish were representative of species living in marine, freshwater and estuarine environments.The range of glycoproteins identified in these fish was similar to that found in mammalian tissue in that both neutral and acid glycoproteins were present, the latter included both sialomucins sensitive and resistant to sialidase, and sulphomucin. A single goblet cell contained either neutral or acid glycoproteins alone or in combination. Only the epidermis of the plaice and rainbow trout contained uniform cell populations producing acid glycoproteins, the former sulphomucin and the latter mainly sialomucin. At each site in the flounder and in the gill epithelia of the plaice and rainbow trout, the goblet cell population was mixed, with cells producing each type of glycoprotein. The number of goblet cells producing each type of glycoprotein varied at each tissue site.