INTRODUCTION

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Because a mucosal epithelium is present at the interface with the environment at all internal body surfaces, the mucosa is often the first line of defense against environmental injury and uses mechanisms complementary to those used by lymphocytes (Figure 1). Mucosal cells react to pathogens faster than do lymphocytes and their pathogen-recognition strategy is more general. Although it is difficult to estimate the proportion of infections blocked at the mucosal level versus those that engender a lymphocyte-mediated response, it seems likely from more recent studies that mucosal mechanisms play a crucial role (1). To the extent that the mucosa alone can handle an incipient infection, the host can avoid the occurrence of lymphocyte inflammation and its attendant morbidity. These considerations indicate that understanding and manipulating the innate immune system of the mucosa might, in the context of emerging new pathogens and drug-resistant strains, be extremely beneficial.

View larger version (50K): [in this window] [in a new window]   Figure 1.   Diagram depicting the rapid response of epithelial cells and the delayed response of lymphocytes to a single bacterial pathogen. Whereas epithelial cells secrete preformed defensins, lysozyme, lactoferrin, and mucin shortly after contact with the pathogen, lymphocytes require activation in regional lymph nodes (LN), transit via efferent lymphatics to the heart and peripheral circulation, and homing to the site of bacterial infection.

	HOW MUCOSAL IMMUNITY WORKS

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There is convincing evidence that epithelial cells can detect the presence of pathogens and irritants and respond by altering gene expression. Altered gene expression takes two forms: (1) the upregulation of genes whose products directly attack or clear the contaminant (2, 3), and (2) the upregulation of genes whose products (cytokines) recruit leukocytes (4).

Epithelial gene products aimed at killing bacteria are (1) lysozyme, which lyses bacterial cell walls, (2) lactoferrin, which sequesters iron needed for bacterial metabolism, and (3) defensins, which introduce pores in the cell wall that impair bacterial homeostasis. Mucin, an epithelial glycoprotein, does not kill bacteria, but forms a viscoelastic gel (mucus) that traps bacteria along with other contaminants. In cooperation with epithelial cilia, mucus transports contaminants toward the throat, where they are swallowed and delivered to the gastrointestinal tract for degradation.

It can be inferred from the nonselective nature of the "mucociliary escalator" that mucin has a more general protective function than do lysozyme, lactoferrin, and defensins. Thus, any inhaled contaminant, bacterial or otherwise, that is trapped in the sticky mucous layer will be cleared by mucociliary transport. Consistent with this, clinical studies show that mucin production increases in response not only to pathogens but also to inhaled particles and irritants.

Our interest in the control of mucin production is based on the all too frequently occurring phenomenon in which mucin is not only upregulated but is excessively produced in response to inhaled contaminants. The phrase "too much of a good thing" aptly describes the situation in which infection or irritation engenders so much mucin production as to overwhelm the ability of the fine, hairlike epithelial cilia to move it. When it stagnates, mucus constitutes a favorable niche for bacteria and can add to, rather than alleviate, pathology induced by noxious stimuli. The deleterious effects of overproduction of mucus in human airways motivates us to understand mechanisms underlying this phenomenon and to design effective therapeutic interventions.

Our group's recent forays into the realm of signal transduction pathways controlling mucin transcription have revealed a complex situation in which multiple noxious stimuli induce in epithelial cells the same mucin gene by distinct yet intersecting pathways. For example, both gram-positive and gram-negative bacteria activate MUC 2 transcription via the same mitogen-activated protein (MAP) kinase (Erk 1/2), although the epithelial cell surface receptors and mucin gene response elements used in each case are different. From the results described below it will be evident that noxious environmental stimuli as diverse as air pollution, gram-positive and gram-negative bacteria, tobacco smoke, and lymphocyte mediators all have the capacity to stimulate mucin gene transcription and do so through distinct yet intersecting signaling pathways. The challenge for therapeutic intervention is to understand the intimate details of each pathway in order to design drugs that are effective in inhibiting overproduction of mucus yet do not promiscuously interfere with the myriad other host defense mechanisms that are dependent on the same common signaling molecules.

	HOW DID RELATED BUT NONIDENTICAL SIGNALING PATHWAYS ARISE?

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Many mammalian epithelial proteins are homologs of host defense proteins used by primitive organisms. For example, defensins are produced by Drosophila and mucin is produced by snails and hagfish. Even more ancient in evolution than the proteins themselves, however, are the signaling pathways controlling their synthesis. Remarkably, studies of plant genes involved in pathogen resistance (R genes) show that some of these are similar to pathogen resistance genes in insects and mammals (5). One set of plant genes encodes proteins that are similar to mammalian cell surface receptors. For example, the tobacco N gene shows homology to genes encoding both the mammalian interleukin (IL)-1 receptor and the Drosophila Toll family of receptors, all of which are involved in host defense (6). Another set of plant genes, including the tomato pto, pti, and fen genes, encode cytoplasmic kinases homologous to the Toll-associated kinase Pelle and the IL-1 receptor-associated kinase IRAK (7). The presence of homology between plant and animal genes indicates that these genes derive from ancestral genes present in organisms that lived before the bifurcation between plant and animal phyla (1 billion years ago). It has been speculated that the "original" pathogen resistance pathway contained an IL-1 receptor-like protein with an intracellular kinase domain to convey signals to an NF-B-like transcription factor and thence to the promoter of an ancient pathogen resistance gene. If so, as previously suggested (10), 1 billion years of evolution could have permitted mutations leading to the genesis of a cytoplasmic kinase such as IRAK that took over the signaling function of the receptor cytoplasmic domain, thereby allowing mutation of the ectodomain to respond to other noxious stimuli. Other such mutations could have provided additional versatility.

Although hypothetical, a scenario like the one described above could explain the nest of intersecting cell stress pathways present in the epithelial cells of modern mammals. While this idea does not simplify the job of unraveling the pathways themselves, it does reassure us that when we examine signaling triggered by stimuli as diverse as bacteria and tobacco smoke, and consistently find dependence on molecules such as MAP kinase and NF-B, we are probably on the right track.

	STRATEGY FOR UNDERSTANDING PATHWAYS CONTROLLING MUCIN PRODUCTION

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From the work of Reid and colleagues, showing increases in the number and size of periodic acid-Schiff-positive airway epithelial cells in response to noxious stimuli (11), it seemed likely that the respiratory epithelium produces a fixed amount of mucin at baseline and in addition contains mechanisms permitting mucin upregulation in case of "emergency." Our studies confirm that this is true (3, 16). By modeling noxious environmental stimuli in vitro, we showed that each stimulus we applied (supernatant from gram-negative and gram-positive bacteria, lipopolysaccharide, tobacco smoke, the air pollutant residual oil fly ash [ROFA], and lymphocyte mediators) caused increases in the production of mucin mRNA by epithelial cells. In all cases, this is controlled at the level of RNA transcription.

To decipher stimulus-specific control mechanisms, we use a bioassay consisting of airway epithelial cells in culture (Figure 2). The cells are transfected with a chimeric gene consisting of a luciferase reporter gene driven by a fragment of the mucin gene promoter. To examine the response of the transfected cells to various environmental threats, we have modeled as closely as possible the natural environmental stimuli associated with bacteria, tobacco smoke, allergy-associated leukocytes, and air pollution. We routinely measure stimulus-induced luciferase reporter gene activity in epithelial cells as an experimental end point. We express the ratio between mucin promoter activity induced by a noxious stimulus and that present in the absence of stimulus. The higher the ratio, the greater the extent to which a given stimulus activates mucin transcription. Results acquired in this manner are evaluated for authenticity by monitoring changes in expression of the endogenous mucin gene induced by the same stimulus.

View larger version (70K): [in this window] [in a new window]   Figure 2.   Diagram depicting the experimental system and strategy used to study mucin transcription in response to bacterial stimuli. Bacterial exoproducts within the supernatant of bacteria grown to late log phase are applied to human epithelial cells in culture. The human cells have been transfected with a luciferase reporter gene under the control of a mucin (MUC 2) promoter. Hours later, epithelial cell lysates are prepared for luciferase assay. Typically, cells treated with bacterial supernatant (Bacterial Sup) show elevated levels of activity compared with cells cultured under control (Con) conditions. The intensity of the mucin response to bacteria can be modulated by drugs specifically blocking relevant signaling molecules. t.f. = transcription factor.

	ACTIVATION OF MUCIN TRANSCRIPTION BY GRAM-NEGATIVE BACTERIA (Pseudomonas aeruginosa)

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We originally focused on the possibility that Pseudomonas aeruginosa, a virulent bacterium that colonizes the lungs of patients with cystic fibrosis, is capable of stimulating mucin transcription in lung epithelial cells. By using deletion mutants of the MUC 2 mucin promoter in transient transfections of lung and colon carcinoma cells, we identified two P. aeruginosa-sensitive enhancers. By gel and supershift assays, we determined that the more distal enhancer (~ 1.5 kb) mediates P. aeruginosa-induced MUC 2 transcription via the binding of NF-B (p50 and p65 subunits) to the B site upstream of MUC 2. The identity of the protein binding to the proximal enhancer (343/73) is unknown at this time. Through the use of chemical inhibitors and dominant negative mutants, we determined that P. aeruginosa exoproducts activate NF-B via a Src-Ras-MAPK-pp90 RSK signaling pathway (3, 19).

	ACTIVATION OF MUCIN TRANSCRIPTION BY GRAM-POSITIVE BACTERIA (Staphylococcus aureus)

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We have reported that gram-positive as well as gram-negative bacteria increase steady state levels of MUC 2 and MUC 5 AC mRNA (18). Our studies further indicate that this upregulation is controlled at the level of transcription. This permits use of the luciferase reporter gene assay to decipher some of the signaling pathways involved as well as to examine the role of specific DNA elements in the response. In studies so far, we have defined a major gram-positive activity and are examining the possibility that this acts through the platelet-activating factor (PAF) receptor (20, 21). Beyond that, we have seen that mucin induction by gram-positive bacteria, like induction by gram-negative bacteria, is tyrosine kinase dependent (18) and requires the signaling kinase MEK 1/2, implying the involvement of the MAP kinase Erk 1/2. Although evidence for the involvement of MEK 1/2 seemed to suggest that MUC 2 induction by gram-negative and -positive organisms might converge at this level, this does not seem to be the case: the principal gram-positive MUC 2 response element is significantly upstream of that principally responsible for the response to gram-negative bacteria and does not contain a B site.

	ACTIVATION OF MUCIN TRANSCRIPTION BY THE AIR POLLUTANT ROFA

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Epidemiological and experimental studies show that components of air pollution are associated with hypersecretion of mucus (22, 23). We examined the possibility that a specific component of air pollution, the small particle (< 10 µm) termed ROFA (residual oil fly ash), might upregulate mucin gene expression. Results showed that after exposure to ROFA in culture medium, airway epithelial cells did show upregulation of mucin transcription (gene MUC 5 AC). Vanadium, a metal making up to ~ 19% of ROFA by weight, mimicked this effect.

A screen of signaling inhibitors showed that the induction of MUC 5 AC by ROFA was dependent on the MAPK Erk 1/2, the protein tyrosine kinase c-Src, and the GTP-binding protein Ras. This renders it similar to the signaling pathway mediating MUC 2 induction by P. aeruginosa but differs from that pathway in that it is also sensitive to protein kinase C (PKC) inhibitors. Notably, the ROFA studies do not focus on MUC 2 but on another mucin family member, MUC 5 AC. It is clear that although the promoters of MUC 2 and MUC 5 AC differ substantially in sequence they are functionally homologous with respect to their sensitivity to at least some intracellular signals.

The fact that vanadium is a potent phosphatase inhibitor may indicate that effects of the air pollutant ROFA on mucin and potentially other host defense genes are exerted by the unmasking of phosphorylation-dependent signaling pathways normally associated with pathogen resistance.

	ACTIVATION OF MUCIN TRANSCRIPTION BY TOBACCO SMOKE

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Tobacco smoke causes chronic bronchitis, a disease in which lung epithelial cells overproduce mucin to the extent that it clogs air passages, compromises respiration, and predisposes the lung to infection. We have observed that smoke is capable of directly stimulating mucin transcription in epithelial cells. The smoke component triggering the response as well as the epithelial surface signaling events remain obscure at this time. It is interesting to note, however, that smoke represents yet another noxious stimulus inducing mucin transcription through c-Src and MEK 1/2 but that unlike the pathway induced by P. aeruginosa, it does not culminate in NF-B activation.

	ACTIVATION OF MUCIN TRANSCRIPTION BY ALLERGIC AIRWAY FLUID

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On the basis of reports that patients with asthma show airway hypersecretion of mucus, we hypothesized that there might exist factors in the airway lining fluid that stimulate mucin synthesis. We obtained (in collaboration with J. Fahy and H. Boushey, University of California, San Francisco) aspirates from the airways of six patients with asthma and six unaffected individuals. Whereas the fluid from control airways caused a modest increase in mucin transcription, that from asthmatic airways caused a much greater increase (24). To identify both the activity present in airway fluid and signal transduction mechanisms in the epithelial cells, we turned (in collaboration with D. Bice of the Lovelace Inhalation Toxicology Institute, Albuquerque, NM) to an animal model of allergic airway disease generated in dogs. Dogs were sensitized with ragweed during the first weeks of life and challenged intrabronchially at variable intervals thereafter. In comparing the potency of bronchoalveolar lavage (BAL) fluid before and after ragweed challenge, we found, as in human airway fluid, that the prechallenge BAL stimulated mucin transcription modestly and that the postchallenge BAL stimulated transcription more strongly (24). In efforts to identify the promucin activity in BAL fluid, we subjected it to size fractionation followed by bioassay of fractions in the mucin reporter assay. We identified activity in fractions containing molecules 30- 100 and < 10 kD in size. On the basis of previous studies showing that activated helper T cell type 2 (Th2) but not Th1 lymphocytes and intratracheal ovalbumin evoked mucous cell metaplasia in mouse airways (25) we hypothesized that a Th2 mediator, 30-100 or < 10 kD in size, accounted for the ability of allergic airway fluid to upregulate mucin transcription. Th2 cytokines in these size ranges include IL-5, IL-9, and IL-13. In attempts to block the stimulatory effect of allergic BAL fluid with antibodies inhibiting the interaction between IL-5, IL-9, and IL-13 and their receptors, we observed that IL-9 but not IL-5 or IL-13 is required for the response (24). We subsequently showed that IL-9 but not IL-5 or IL-13 is capable of stimulating MUC 5 AC transcription in our luciferase reporter assay.

These studies implicate IL-9 and its receptor in allergic hypersecretion of mucus. Although we have not yet tested the effects of broad-spectrum signaling inhibitors on the downstream signaling pathway, previous studies of IL-9 signaling in lymphocytes (26) strongly suggest that the response is mediated by various components of the JAK-STAT signaling pathway. Thus, epithelial cells may have one set of receptors and signaling molecules with which they respond to pathogens and another with which they respond to lymphocyte mediators.

	CONCLUSION

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The availability of promoter assays make it possible to efficiently dissect signaling pathways that mediate the effects of diverse insults to host cells. Results indicate that each type of stimulus (bacterium, pollutant, smoke, lymphocyte mediator) acts through a distinct cell surface receptor, a distinct signaling pathway, and distinct gene regulatory elements to activate transcription of even a single mucin gene. Despite this diversity, common elements are emerging. Among these are c-Src, the MAP kinase kinase MEK 1/2, and NF-B. Therapeutic strategies will have to balance the advantages of using a broad- spectrum inhibitor that could attenuate hypersecretion of mucus of diverse etiologies against the advantages of a narrow-spectrum inhibitor that would leave as intact as possible the injury response system of the patient.