Regulation of mucous differentiation and mucin gene expression in the tracheobronchial epithelium.

The goal of our studies is to elucidate mechanisms that control and modulate mucous differentiation and mucin gene expression in the conducting airways. We used cultures of normal human tracheobronchial epithelial (NHTBE) cells that were shown to secrete two major airway mucins, namely MUC5AC and MUC5B as well as several other secretory products. Mucous differentiation and expression of MUC2, MUC5AC, MUC5B and MUC7, but not MUCi, MUC4, and MUC8 mucin genes, were shown to be retinoic acid- (RA) or retinol-dependent. We found that RA control of mucin genes was mediated by the retinoid acid receptors RAR alpha and, to a lesser extent, by RAR gamma. Our studies also showed that other important bioregulators such as thyroid hormone (T3) and epidermal growth factor (EGF) modulate basal expression of mucin genes, interacting with RA in a concentration-dependent manner. T3, which binds to thyroid receptors (TRs) belonging to the same superfamily of steroid hormone nuclear receptors as the RARs, inhibits mucin gene expression, particularly MUC5AC. One possible mechanism of this T3 effect is downregulation of RAR proteins, which are critical for mucin gene expression. However, we also found that T3 inhibits MUC5AC transcription.EGF, which had previously been shown to stimulate mucin expression and mucin secretion in cultured rat tracheal epithelial (RTE) cells, inhibited mucin secretion in human bronchial epithelial cell cultures. This effect was EGF concentration- and time-dependent and was progressively abolished by increasing the RA concentration. Subsequent studies suggested that the inhibitory effects of high concentrations of EGF may result from selective reduction of MUC5AC expression. These studies thus point to potentially important species differences in the mechanisms regulating mucous production, and they also confirm previous findings indicating differential regulation of MUC5AC and MUC5B gene expression.

Regulation of mucin gene expression in human tracheobronchial epithelial cells by thyroid hormone.

We reported previously that the expression of the gene encoding MUC5AC mucin in human airway epithelial cells is controlled by retinoic acid via the retinoic acid receptor (RAR)-alpha and that 3,3',5-tri-iodothyronine (T(3)) inhibits the expression of MUC5AC. The purpose of the present study was to identify mechanisms mediating the effect of T(3). T(3) has been shown to inhibit gene expression via several mechanisms, either by enhancing or repressing the transcription of target genes or by the regulation of post-transcriptional events. Results showed that T(3) strongly inhibited MUC5AC-driven luciferase activity in normal human tracheobronchial epithelial cells that had been transiently transfected with a MUC5AC-luciferase reporter construct; however, it did not affect MUC5AC mRNA stability. These results indicate that T(3) suppresses MUC5AC expression at the transcriptional level. An analysis of deletion constructs showed that deletion of the region downstream of 3 kb resulted in markedly decreased levels of MUC5AC transcription in the absence of T(3) (i.e. under control conditions) as well as a loss of responsiveness to the inhibitory effects of T(3). This suggests that this region might contain elements important for the activation as well as the repression of MUC5AC transcription. To determine whether T(3) modulates retinoic-acid-dependent MUC5AC transcription via an alteration in the abundance of retinoid receptor proteins, we examined the type and abundance of these receptors in nuclear extracts of airway epithelial cells grown in the presence or absence of T(3). Western blots showed that T(3) markedly decreased several types of retinoid receptor while not affecting T(3) receptor proteins. Consistent with this finding were gel-shift assays revealing a decrease in RAR-retinoic acid response element complexes obtained from T(3)-treated cells. We propose that T(3) might inhibit retinoid-dependent MUC5AC expression by decreasing retinoid receptor levels and thereby decreasing the transcriptional activation of this gene for mucins.

Regulation of differentiation of the tracheobronchial epithelium.

The study of differentiation has been the domain of embryologists and developmental biologists and, in the pulmonary field, the concern of neonatologists. Why should those of us who are neither be interested in differentiation of the epithelium lining the conducting airways? The reason is that injury to the airway epithelium and disruption of its steady state and its normal differentiation are common occurrences in both acute episodes of infection and during chronic diseases such as chronic obstructive pulmonary disease and asthma. Thus, it is important to know how injury is repaired and which are the critical mechanisms that control and regulate differentiation.

KPL1, which encodes a novel PH domain-containing protein, is induced during ciliated cell differentiation of rat tracheal epithelial cells.

Using differential display, we have identified a novel gene, KPL1, induced in rat tracheal epithelial (RTE) cells grown under conditions which stimulate ciliogenesis. The KPL1 protein is predicted to contain a pleckstrin homology (PH) domain, which has been found in numerous signal transduction and cytoskeletal proteins. These domains are thought to function by recruiting proteins to cellular membranes, and they have been shown to bind phosphoinositols and the beta/gamma subunit of G proteins. We have cloned rat and human KPL1; the predicted protein translations are 94% identical. Alternate transcripts exist in rat and human tracheal cells that predict a protein which contains a 35-amino acid insert. KPL1 was upregulated in RTE cultures undergoing mucociliary but not squamous differentiation; and in cultures undergoing mucociliary differentiation, KPL1 expression most closely paralleled that of a marker of ciliated cell differentiation (axonemal dynein heavy chain) and not a marker of mucous cell differentiation (mucin 5AC). As a new member of the family of PH domain-containing proteins, KPL1 may have a unique role in ciliated cell differentiation or function.

Characterization of mucins from cultured normal human tracheobronchial epithelial cells.

Early-passage normal human tracheobronchial epithelial (NHTBE) cells grown in air-liquid interface cultures in medium containing retinoids differentiate into a mucociliary epithelium over a 2- to 3-wk period and express increasing mRNA levels of the airway mucin genes MUC5AC and MUC5B as the cultures age; the levels of MUC2 mRNA were very low throughout the study. Using specific antibodies to MUC5AC and MUC5B mucins, we noted a gradual increase in these two mucins in the intracellular and apically secreted pools as a function of time. A low level of MUC2 mucin was detected, which did not change with time. The intracellular and apically secreted mucins isolated from day 14 and day 21 cultures by density gradient centrifugation were similar in density to those previously isolated from human respiratory mucus secretions. The sedimentation rate of the apically secreted mucins indicated that they were highly oligomerized, polydisperse macromolecules similar to those previously documented from in vivo secretions. In contrast, the cell-associated mucins from the cultured NHTBE cells were much smaller, possibly only monomers and dimers. Anion-exchange chromatography detected no differences in charge density between the reduced and carboxymethylated cell-associated and secreted forms of the MUC5AC and MUC5B mucins. The MUC5AC mucin was of similar charge density to its in vivo counterpart; however, MUC5B was more homogeneous than that found in vivo. Finally, evidence is presented for an intracellular NH(2)-terminal cleavage of the MUC5B mucins. These studies indicate that the mucins produced by cultured NHTBE cells are similar to those found in human airways, suggesting that this cell culture model is suited for studies of respiratory mucin biosynthesis, processing, and assembly.

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.

Lysozyme expression during metaplastic squamous differentiation of retinoic acid-deficient human tracheobronchial epithelial cells.

We previously reported (Gray, T. E., K. Guzman, C. W. Davis, L. H. Abdullah, and P. Nettesheim. 1996. Mucociliary differentiation of serially passaged normal human tracheobronchial epithelial cells. Am. J. Respir. Cell Mol. Biol. 14:104-112) that retinoic acid (RA)-deprived cultures of normal human tracheobronchial epithelial (NHTBE) cells became squamous, failed to produce mucin, and instead secreted or released large amounts of lysozyme (LZ). The purpose of the studies reported here was to elucidate the relationship between RA deficiency-induced squamous differentiation and increased LZ, and to determine what mechanisms were involved. We found that intracellular LZ began to accumulate in RA-deficient NHTBE cultures early during squamous differentiation. Between Days 10 and 18 of culture, cellular LZ levels were more than 10 times higher in RA-deficient than in RA-sufficient cultures. On Day 12, large numbers of cells began to exfoliate in RA-deficient cultures and extracellular LZ appeared at the apical surface, presumably released from the exfoliated cells. Metabolic labeling studies showed that the rate of LZ synthesis was not increased in RA-deficient cultures over that in RA-sufficient cultures; however, intracellular LZ half-life was much longer in RA-deficient cultures. We concluded that the increased accumulation of both intra- and extracellular LZ in RA-deficient cultures was due to increased LZ stability and was not the result of increased LZ synthesis. When RA-deficient cultures were treated on Day 7 with 10(-6) M RA, intracellular LZ levels did not substantially decrease until 3 d later, coinciding with a marked increase in mucin secretion. LZ messenger RNA levels were unchanged at 24 h, but were modestly increased (rather than decreased) at all subsequent time points. We concluded that RA does not directly regulate LZ, and that the excessive accumulation of LZ in RA-deprived NHTBE cells is a consequence of vitamin A deficiency-induced abnormal differentiation.

Cloning and characterization of KPL2, a novel gene induced during ciliogenesis of tracheal epithelial cells.

To identify genes upregulated during the process of ciliated cell differentiation of airway epithelial cells, differential display was used to compare RNA from rat tracheal epithelial (RTE) cells cultured under conditions that inhibit/promote ciliated cell differentiation. Several partial complementary DNAs (cDNAs) were identified whose expression was regulated coordinately with ciliated cell differentiation. One of these, KPL2, detected a messenger RNA transcript of approximately 6 kb when used as a probe on Northern blots of RNA from ciliated cultures but was undetectable in RNA from nonciliated cultures. Sequencing of overlapping clones obtained by a modified rapid amplification of cDNA ends procedure generated a complete cDNA sequence that exhibited no significant homology to sequences in GenBank, indicating that KPL2 is a novel gene. Southern analysis demonstrated that KPL2 exists as a single-copy gene. KPL2 contains a long open reading frame predicted to code for a protein of > 200 kD. Several putative functional motifs are present in the protein, including a calponin homology domain, three nuclear localization signals, a consensus P-loop, and a proline-rich region, suggesting that KPL2 has a unique function. KPL2 was undetectable in heart and liver samples, but was expressed in brain and testis, tissues that contain axonemal structures. In seminiferous tubules of the testis, KPL2 expression was stage-specific and appeared to be highest in spermatocytes and round spermatids. During differentiation of RTE cells, the expression of KPL2 closely paralleled that of an axonemal dynein heavy chain. These results suggest that KPL2 plays an important role in the differentiation or function of ciliated cells in the airway.

Role of retinoid receptors in the regulation of mucin gene expression by retinoic acid in human tracheobronchial epithelial cells.

To investigate which retinoid receptors are critical in the regulation by all-trans-retinoic acid (RA) of the mucin genes MUC2, MUC5AC and MUC5B in cultured normal human tracheobronchial epithelial (NHTBE) cells, we used pan-RAR-, pan-RXR- and RAR- isotype (alpha, beta and gamma)-selective agonists and RARalpha- and RARgamma-selective antagonists (RAR is RA receptor and RXR is retinoid X receptor). RAR-, RARalpha- and RARgamma-selective agonists strongly induced mucin mRNAs in a dose-dependent manner, while the RARbeta-selective retinoid only weakly induced mucin gene expression at very high concentrations (1 microM). The pan-RXR-selective agonist by itself did not induce mucin gene expression, but acted synergistically with suboptimal concentrations of the pan-RAR agonist. A retinoid with selective anti-activator-protein-1 activity only marginally induced mucin gene expression. The RARalpha antagonist strongly inhibited mucin gene induction and mucous cell differentiation caused by RA and by the RARalpha- and RARgamma-selective retinoids. In contrast, the RARgamma antagonist only weakly inhibited RARalpha-selective-retinoid-induced mucin gene expression, but completely blocked mucin gene expression induced by the RARgamma-selective retinoid. Our studies indicate that RARalpha is the major retinoid receptor subtype mediating RA-dependent mucin gene expression and mucous cell differentiation, but that the RARgamma isotype can also induce mucin genes. Furthermore these studies suggest that RARbeta is probably not (directly) involved in RA-induced mucin gene expression.

Restoration of the mucous phenotype by retinoic acid in retinoid-deficient human bronchial cell cultures: changes in mucin gene expression.

Retinoid-deficient cultures of airway epithelial cells undergo squamous differentiation. Treatment of such cultures with retinoic acid (RA) leads to restoration of the mucous phenotype. The purpose of our study was to characterize the cellular and molecular changes following RA treatment of retinoid-deficient human tracheobronchial epithelial cell cultures. Of particular interest was to determine when during the conversion of the squamous to the mucous phenotype the mucin genes MUC2, MUC5AC, and MUC5B were expressed. We used cornifin alpha and secreted mucin as markers to monitor the squamous and mucous phenotypes, respectively. Our studies showed that the RA responsiveness of the cultures progressively decreased with protracted retinoid deficiency, requiring higher RA concentrations to restore the mucous phenotype. Within 12 h after the start of RA treatment, cornifin alpha expression decreased, signaling the beginning of a change in cellular phenotype. At 24 h after addition of RA to the cultures, a significant number of mucous cells appeared, and at 72 h mucin was secreted in measurable amounts. Induction of mucin gene expression occurred sequentially: MUC2, MUC5AC, and MUC5B mRNAs were upregulated at 24, 48, and 72 h, respectively. When cultures maintained in 10(-8) M RA were treated with 10(-6) M RA, MUC2 but not MUC5AC and MUC5B mRNA levels were upregulated within 6 h. Our study indicates that MUC2 mRNA is an early marker of mucous differentiation, whereas MUC5AC and MUC5B mRNAs are expressed during more advanced stages of mucous differentiation. Our studies further suggest that each of the mucin genes is regulated by distinct mechanisms.

Differentiation dependency of eicosanoid enzyme expression in human tracheobronchial cells.

The expression of 15-lipoxygenase (15-LO) and prostaglandin H synthase-2 (PGHS-2) was studied in retinoic acid (RA) sufficient and RA-deficient normal human tracheobronchial epithelial (NHTBE) cell cultures. In the absence of RA, in which the cultures became squamous metaplastic, neither 15-LO nor PGHS-2 were expressed. In RA-sufficient cultures, which differentiated into a mucociliary epithelium, both enzymes were expressed: PGHS-2 during early phases and 15-LO during late stages of differentiation. Depending on the stage of differentiation, the RA-sufficient cultures produced PGE, and in the presence of exogenous linoleic acid (LA) 13-HODE. Experiments are underway to examine the effects of inflammatory cytokines on eicosanoid metabolism and the role these metabolites play in airway diseases.

Differentiation-dependent regulation of retinal dehydrogenase gene expression in the trachea.

Retinoic acid (RA), a metabolite of vitamin A, is known to be a key signaling molecule in regulating epithelial cell differentiation. We recently characterized and cloned a retinal dehydrogenase (RALDH) that catalyzes the oxidation of retinal to RA. In this study, we investigated the effects of retinoids on the level of RALDH mRNA and protein as well as RALDH activity in the trachea and cultured tracheal epithelial cells. Vitamin A deficiency induced squamous metaplasia in the tracheal epithelium and down-regulated RALDH expression. Supplementation of retinol and retinoic acid to vitamin A deficient rats restored the normal mucociliary epithelium and up-regulated the RALDH expression. In rat epithelial cells cultured in vitro, RAinhibited squamous differentiation and promoted mucociliary differentiation. Squamous differentiated cultures (RA-) expressed very low levels of RALDH mRNA, whereas mucociliary differentiated cultures (RA+) expressed high levels of RALDH mRNA. Retinal and retinol were poor inducers of mucociliary differentiation as well as RALDH expression. The RALDH expression paralleled the expression of the mucin-1 gene in mucociliary cultures. These results suggest that the expression of RALDH is dependent on the differentiation state of the airway epithelium.

Expression of nitric oxide synthase isoforms in normal human tracheobronchial epithelial cells in vitro: dependence on retinoic acid and the state of differentiation.

The retinoic acid (RA) and differentiation dependence of constitutive expression of the nitric oxide synthase (NOS) isoforms, iNOS, eNOS, and bNOS, was examined by reverse transcriptase polymerase chain recitation (RT-PCR) in cultured, normal, human, tracheobronchial epithelial (NHTBE) cells. In the presence of RA (RA+), early passage NHTBE cells grown in air-liquid interface (ALI) cultures undergo mucous differentiation; in the absence of RA (RA-), they undergo metaplastic squamous differentiation. Under both conditions the respective differentiated phenotype develops around day 10 of culture. We found that iNOS mRNA levels were much higher in RA+ cultures, expressing the mucous phenotype, than in RA- cultures, expressing the metaplastic squamous phenotype. In contrast, eNOS mRNA levels were much higher in RA- cultures than in RA+ cultures. Expression of bNOS was not significantly affected by the RA status. The pattern of expression of NOS isoforms was then studied during the course of development of the two cellular phenotypes. During the early stages of differentiation, expression of iNOS (RA+) and eNOS (RA-) was very low, indicating that the expression of these two isoforms was not only dependent on the presence or absence of RA, but also on the degree of differentiation. The differentiation dependence of bNOS mRNA was less obvious. Four days of RA treatment of RA- cultures, which reverses the squamous phenotype and restores mucous differentiation, induced iNOS expression in a concentration-dependent manner. eNOS expression was depressed by 10(-8) M RA, while bNOS mRNA levels were slightly reduced by 10(-6) M RA. No NOS proteins were detected in unstimulated RA+ and RA- cultures. iNOS protein was induced by cytokine treatment in RA+ cultures, in contrast to eNOS and bNOS protein levels, which were unaffected. Our studies show that constitutive expression of the NOS isoforms is differentially regulated and that iNOS and eNOS mRNA levels are dependent on the stage of mucous and squamous differentiation, respectively. bNOS expression was only marginally affected by the RA or differentiation status.

Changes in expression of 15-lipoxygenase and prostaglandin-H synthase during differentiation of human tracheobronchial epithelial cells.

The purpose of our studies was to examine differentiation-dependent expression of 15-lipoxygenase (15-LO) and prostaglandin H synthase (PGHS) isoforms in cultured normal human tracheobronchial epithelial cells. In the presence of retinoic acid (RA) the cultures differentiated into a mucociliary epithelium. When cultured in RA-depleted media, the cultures differentiated into a squamous epithelium. In the absence of RA the cultures did not express 15-LO or either of the PGHS isoforms. The PGHS-1 isoform was not expressed in RA-sufficient cultures, but both PGHS-2 messenger RNA (mRNA) and protein were strongly expressed, and prostaglandin E2 (PGE2) was produced during the predifferentiation phase. No PGHS-2 expression or PGE2 could be detected in fully differentiated mucociliary cultures. 15-LO showed the opposite expression pattern: neither mRNA nor protein were detected during the predifferentiation stage, but both were strongly expressed once mucous differentiation had occurred. Cytosolic phospholipase A2 protein was expressed throughout all stages of growth and differentiation. The cultures generated no 15-LO metabolites when incubated with 10 microM to 50 microM arachidonic acid (AA) and stimulated with ionophore. However, lysates prepared from such cultures generated 15-hydroxyeicosatetraenoic acid (15-HETE) and 12-HETE from AA, indicating that the cells contained active enzyme. When cultures expressing 15-LO protein were incubated with 10 microM linoleic acid (LA) instead of AA, and were stimulated with ionophore, they generated 13-hydroxy-9,11-octadecadienoic acid. LA rather than AA appeared to be the preferred substrate for the 15-LO enzyme. Our studies indicated that the expression of 15-LO and PGHS-2 is differentiation dependent in airway epithelial cells.

Expression and regulation of gamma-glutamyl transpeptidase-related enzyme in tracheal cells.

Glutathione plays an essential role in protecting the pulmonary system from toxic insults. gamma-Glutamyl transpeptidase-related enzyme (GGT-rel) is a novel protein capable of cleaving the gamma-glutamyl peptide bond of glutathione and of converting leukotriene C4 to leukotriene D4. A rat homologue of GGT-rel was identified and was found to be highly expressed in cultures of differentiating rat tracheal epithelial (RTE) cells. The 2.6-kb cDNA predicts a 572-amino acid protein with 79% identity to human GGT-rel. GGT-rel was weakly expressed in normal trachea but was strongly induced by epidermal growth factor in cultures of RTE cells. GGT-rel was also highly expressed in lung tumors induced by inhalation of isobutyl nitrite. These results demonstrate that GGT-rel 1) is expressed in normal tracheal cells, 2) can be induced by epidermal growth factor, and 3) is elevated after chemical exposure. The induction of high levels of GGT-rel may play an important role in protecting the lung from oxidative stress or other toxic insults.

Regulation of the secretory phenotype of human airway epithelium by retinoic acid, triiodothyronine, and extracellular matrix.

The purpose of our studies was to identify factors which regulate the composition of airway secretions produced by normal human tracheobronchial epithelial (NHTBE) cells. Individual factors were removed from the culture media of NHTBE cells grown in air-liquid interface (ALI) cultures (which support mucociliary differentiation) and the effects on mucin, lysozyme (LZ), and secretory leukocyte protease inhibitor (SLPI) secretion and gene expression were examined. Deletion of hydrocortisone, epinephrine, transferrin, or gentamycin-amphotericin from the media had no reproducible effects; deletion of insulin was incompatible with culture growth. We identified 3 factors, namely retinoic acid (RA), triiodothyronine (T3) and collagen gel substratum, which had a major impact on the profile of NHTBE secretions. Removal of RA from the media caused a drastic decrease in mucin secretion and a decrease in expression of the mucin genes MUC2 and MUC5AC.LZ and SLPI secretions were increased in these cultures. Paradoxically LZ mRNA was decreased, while SLPI mRNA levels were increased. Removal of T3 selectively increased mucin secretion, MUC2 gene expression was not affected, but MUC5AC mRNA levels reproducibly increased, suggesting that the expression of these two mucin genes is differentially regulated. LZ and SLPI secretion levels were not significantly affected by deletion of T3 from the culture media; however, LZ mRNA levels were increased in the absence of T3 while SLPI transcript levels were not affected. Omission of the attachment substratum, type I collagen gel, resulted in significant increases in all 3 secretory products. MUC2 and MUC5AC steady state mRNA levels were not consistently affected. In contrast LZ and SLPI gene expression were reproducibly increased. Our studies show that individual factors in the epithelial environment can regulate expression of specific secretory cell gene products in a highly selective manner.

Quantitation of mucin RNA by PCR reveals induction of both MUC2 and MUC5AC mRNA levels by retinoids.

The polydispersity of most human secretory mucin messages has made them difficult to detect specifically and quantitatively, impeding the evaluation of the relative expression of the various mucin genes and their role in normal and pathological conditions. For this reason, we developed competitive reverse transcriptase-polymerase chain reaction (PCR) methods to measure the airway mucins MUC2 and MUC5AC. Oligonucleotide pairs were designed that specifically detect MUC2 and MUC5AC, as demonstrated by the size and sequence of the PCR product and the expected tissue distribution. The mucin oligonucleotide primers were used to synthesize internal competitive standards, called MIMIC. Using this assay, the relative expression of these messages was analyzed in retinoid-replete or -deprived cultures of normal human tracheobronchial epithelial (NHTBE) cells. Retinoid deficiency induces squamous metaplasia in vivo and in vitro. Consistent with these observations and in contrast to a previous report, retinoid-deprived cultures produced at least an order of magnitude less MUC2 and MUC5AC message than retinoid-replete cultures. In summary, this paper describes methodology that can be applied to the specific and quantitative measurement of mucin messages and demonstrates that, in NHTBE cells, the level of MUC2 and MUC5AC mRNA is increased by retinoids.

Tumor necrosis factor alpha stimulates arachidonic acid metabolisms and mucus production in rat tracheal epithelial cell cultures.

Air-liquid interface (ALI) cultures of rat tracheal epithelial (RTE) cells were used to study the response of differentiated airway epithelial cells to the inflammatory cytokine, tumor necrosis factor alpha (TNF alpha). We found that the cultures expressed low levels of TNF alpha receptors. TNF alpha stimulated the AA-cascade: cytoplasmic phospholipase A2 (cPLA2) and prostaglandin H synthase 2 (PGHS2) were upregulated; as a result prostaglandin E (PGE2) secretion was increased. Subsequent to the increase in PGE2 mucus secretion increased, suggesting that PGE2 may act as an autocrine regulator of mucus secretion.

Tumor necrosis factor-alpha modulates the expression of its p60 receptor and several cytokines in rat tracheal epithelial cells.

The epithelium of the conducting airways is frequently the target of toxic chemical and microbial agents causing inflammation, hypersecretion, and epithelial necrosis. TNF-alpha is a prototypical inflammatory cytokine released by macrophages and other inflammatory cells. The purpose of this study was to characterize TNF-alpha receptors in fully differentiated rat tracheal epithelial (RTE) cells in culture and to examine the effects of TNF-alpha on this epithelium. We demonstrated the presence of approximately 250 TNF-alpha receptors per RTE cell. Both known receptor types, p60 (TNF-RI, CD 120a) and p80 (TNF-RII, CD 120b), were expressed. The level of p80 mRNA was unaffected by TNF-alpha treatment, whereas p60 mRNA was down-regulated, and soluble TNF-RI was shed from cells within 30 min. Treatment of RTE cultures with TNF-alpha (1000 U) caused no cytotoxicity (as determined by lactate dehydrogenase release). However, TNF-alpha exposure of the cultures induced the expression of several inflammatory mediators, as determined by reverse transcription-PCR and ELISA. Low levels of IFN-gamma mRNA became detectable after 4 h. Increased levels of TNF-alpha mRNA and protein were found, which peaked after 6 h of TNF-alpha treatment, but neither IL-1 alpha nor IL-1 beta was detectable. Calcium-independent nitric oxide synthase transcripts were elevated two- to threefold within 2 to 6 h of TNF-alpha treatment. These findings suggest that the airway epithelium may actively participate in the pathogenesis of airway inflammation through the production of mediators similar to those found in a Th1 response.