PERSPECTIVE
Trophic Slime, Allergic Slime

Marsha Wills-Karp

Johns Hopkins University, Baltimore, Maryland

The epithelium of the respiratory mucosa provides a barrier against injurious luminal agents, including bacteria, enzymes, and toxins. The normal respiratory epithelium is coated with mucus, which provides a variety of protective functions, including protection of the lower airways from dehydration and from damaging airborne irritants, particles, and microorganisms. The adhesive and viscoelastic properties of mucus glycoproteins (mucins), the major protein components of airway mucus, allow the trapping of foreign substances and their transport and removal on the tips of beating cilia toward the throat, a process termed mucociliary clearance. However, overzealous production of mucus may significantly contribute to the morbidity and mortality associated with certain respiratory diseases. In particular, mucus hypersecretion and plugging of the airways are characteristic features of patients who die from asthma (1, 2), chronic bronchitis, and cystic fibrosis (2).

In human airways, mucins are produced and secreted by specialized cells in the epithelium, including the goblet cells in the surface airway epithelium and the secretory (mucous and serous) cells in the submucosal glands. Because of their greater prominence in histologic sections, submucosal glands, rather than goblet cells, have been thought to contribute the greater quantity of mucus to airway surface fluid (3). However, recent studies suggest that goblet cells may contribute more to the overall quantity of mucus produced than do the submucosal glands. Mucins constitute a heterogeneous group of high molecular weight, richly glycosylated molecules. To date, nine human mucin genes (MUC1, MUC2, MUC3, MUC4, MUC5/5AC, MUC5B, MUC6, MUC7, and MUC8) encoding the protein core of mucin have been identified (reviewed in Reference 4). The biologic importance of these diverse mucin proteins is not currently known. Nonetheless, there appears to be some degree of specificity in the tissue expression of the various MUC genes. In the respiratory tract, seven of the nine MUC genes are expressed (MUC1, MUC5/5AC, MUC2, MUC4, MUC5B, MUC7, and MUC8) (reviewed in Reference 4).

The mechanisms governing mucin glycoprotein synthesis and secretion are not well understood in either health or disease. Control of mucus secretion is a complex process involving regulation at many different levels, including (1) cell proliferation and differentiation, (2) mucin gene expression, and (3) release of mature mucin molecules from storage granules (3). Under normal conditions, there are relatively few goblet cells in the human airway and virtually none in the airways of animals kept in clean environments. In response to a wide variety of stimuli, including proteinases, irritant gases, inflammatory mediators, reactive oxygen species, and cholinergic and nonadrenergic, noncholinergic nerve activation, a rapid increase in the number of airway goblet cells is observed via both hyperplastic (cell division) and metaplastic (cell differentiation) mechanisms. In the airways of rodents, these increases are primarily due to metaplasia, since goblet cells are not normally seen in the airways of clean animals. Secondly, MUC gene transcription has been shown to be induced upon exposure of the airways to a number of substances that induce mucus secretion, such as endotoxin, SO₂, and allergens (5, 6). Thus, it is hypothesized that an important point of control of mucus secretion is the synthesis of these protein backbones of the mature mucin glycoproteins. Although the exact molecular mechanisms regulating mucin-gene expression are virtually unknown, there is evidence that the individual genes may be differentially regulated. For example, Muc-2 expression is induced in the lungs of rats exposed to SO₂ and Sendai virus, but not by allergen exposure (5, 6). Conversely, allergen challenge has been shown to induce MUC5 gene expression (6). Lastly, secretion of mature mucin molecules requires their release from the intracellular granules in which they are stored. To date, there are a number of inflammatory mediators implicated in the allergic diathesis that are known to influence goblet-cell secretion, including the prostaglandins E2 and F2a, leukotrienes, 15-HETES, platelet-activating factor, mast-cell and neutrophil proteases, eosinophil cationic protein, and the cytokines interleukin (IL)-1 and tumor necrosis factor (TNF) (3).

As stated above, mucus hypersecretion is a key feature of allergic asthma and is associated with the clinical symptoms, airway obstruction, and mortality of the disease. Because of the difficulty of studying molecular processes in the human lung, much of our current knowledge of mucus regulation has come from the study of murine models of allergic disease. Recently, several groups of investigators have shown that respiratory challenge with allergens causes physiologic and pathologic changes similar to those seen in human allergic asthma, including airway hyperresponsiveness, airway inflammation, and airway goblet-cell metaplasia (GCM), as evidenced by periodic acid-Schiff (PAS) staining in the airway epithelium (reviewed in Reference 7). More recently, the expression of mucin genes in allergen-challenged mice has been examined (6). Not surprisingly, a particular pattern of mucin-gene expression emerges. Specifically, although other mucin genes such as MUC1 appear to be constitutively expressed in the murine lung, MUC5 expression is markedly upregulated in the lungs of allergen-challenged mice (6). MUC5 gene expression has also been found in lung mucus samples collected from individuals with asthma and from pooled samples from normal individuals (8). In murine models, it is currently thought that GCM and mucus production are directly affected by cytokines that regulate the immune response. Specifically, GCM is associated with a T helper (Th)2 (IL-4, IL-13, IL-5, and IL-9) pattern of cytokine production because adoptive transfer of Th2 cells into the lungs of naive mice results in induction of GCM (9). In sharp contrast, transfer of Th1 cells does not induce similar changes despite the presence of inflammation. The exact mechanisms by which Th2 cytokines induce mucous-cell changes are currently not well understood.

The Th2 cytokine IL-4 was initially implicated in GCM, as overexpression of the IL-4 gene in the airways results in both GCM and Muc-5 gene expression (10). Furthermore, blockade of the IL-4Ra chain in mice sensitized and challenged with antigen abolished airway GCM (11). The importance of the IL-4 signaling pathway was supported by the finding that STAT6-deficient mice were completely devoid of goblet cells both before and after antigen challenge (12). Despite these findings, Cohn and colleagues (9) showed that transfer of Th2 cells lacking the IL-4 gene into the murine lung still resulted in GCM. These data demonstrated that IL-4 was not essential for GCM and highlighted the possibility that other Th2 cytokines were involved in mucous-cell changes in the airways after allergen challenge. In a similar adoptive transfer experiment, Cohn and her colleagues demonstrated that the Th2 cytokine IL-5 was not necessary for GCM (13), although IL-5 transgenic mice have marked GCM (14). This study confirmed previous speculation that eosinophils were not required for mucus hypersecretion. As the IL-4R/STAT6 signaling pathway was implicated, attention turned to the IL-4 look-a-like, IL-13. It was shown that blockade of IL-13 alone in vivo reversed established mucous-cell changes (15, 16). Furthermore, overexpression or administration of recombinant (r)IL-13 in vivo leads to GCM (15, 16, 17) and MUC5 gene expression (16). Together, these studies suggested that although IL-4 likely played a part through its role in Th2 cell differentiation, IL-13 was probably the primary regulator of mucus hyperplasia in vivo. Studies designed to determine whether either IL-4 or IL-13 could directly induce goblet-cell changes have surprisingly shown that neither of these two cytokines induces Muc5 gene expression when administered to epithelial cells in vitro (18, 19). From these studies, we can conclude that the in vivo actions of these cytokines must be due to stimulation of other mediators that directly induce MUC5 gene expression.

Interestingly, in this issue of the Red Journal, Louahed and colleagues (20) demonstrate that the Th2-cell-derived cytokine, IL-9, may be the culprit. They show that IL-9 stimulates GCM and mucin gene expression (Muc-5 and Muc-2) when administered either in vivo or in vitro. They demonstrated in vitro that rIL-9 induced Muc2 and Muc5ac, indicating a direct effect of IL-9 on mucin gene expression. To prove that IL-9 did not induce mucus by induction of other Th2 cytokines, the authors examined message levels of IL-4 and IL-13 in IL-9-treated cells. They showed that IL-9 treatment did not induce either IL-13 or IL-4 message in epithelial cells, suggesting that IL-9 is downstream of these two cytokines. These findings are consistent with a previous report by Longphre and associates (21), who had shown that rIL-9 treatment of epithelial cells in vitro induced Muc5 gene expression. Furthermore, they had shown that antibodies to IL-9, but not to IL-13 or IL-5, inhibited the mucin-stimulating activity of lavage fluids from allergic dogs. In combination, these studies suggest that IL-9 may be the principal Th2 cytokine inducing mucous-cell metaplasia in murine models of allergic disease. However, the mechanism by which IL-9 induces these goblet-cell changes remains a mystery. The IL-9 receptor is composed of the common  chain and the IL-9R chain. Indeed, airway epithelial cells do express the unique IL-9R chain, but it is thought that epithelial cells do not express the common  chain (21). Thus, it is unclear how IL-9 could induce mucous-cell changes directly, unless a unique form of the receptor is expressed. Further, IL-9 receptor signaling is not thought to be mediated via STAT6 activation. Clearly further studies are needed to resolve this issue.

Although IL-9 may be the final common mediator of allergen-induced mucus hypersecretion, the question still remains as to how overexpression of the other Th2 cytokines also results in GCM (Figure 1). There are several possible scenarios to explain the apparent redundancy. First, as alluded to above, IL-4's primary role in vivo is likely the induction of other Th2 cytokines such as IL-9. However, IL-13 and IL-5 are not thought to contribute to Th2-cell differentiation; thus their ability to induce GCM when overexpressed in vivo is still unexplained. It is conceivable that they induce release of IL-9 from non-T cells, such as eosinophils and mast cells. Alternatively, these cytokines may induce mucous-cell changes via indirect mechanisms, which are independent from those induced by IL-9. Our preliminary studies would support this hypothesis, as we have shown that the ability of rIL-13 to induce GCM is dependent on STAT6, suggesting that this signaling pathway is essential for T-cell differentiation, as well as the downstream events leading to mucous-cell hypersecretion (M. Wills-Karp, unpublished data). Lastly, it is possible that each of these cytokines regulates a different step in the cascade of events leading to mucus secretion. This possibility is supported by the reported ability of both IL-13 and IL-4 to induce proliferation of epithelial cells in vitro (22). Although clearly much remains to be learned about the mechanisms by which Th2 cytokines control mucus secretion in allergic asthma, the recent resurgence of interest in this area should fuel a speedy dissection of the exact cellular and molecular mechanisms governing mucus hypersecretion in inflammatory diseases such as asthma.

View larger version (27K): [in this window] [in a new window]   Figure 1.   A hypothetical scheme of the role of Th2 cytokines in mucus hypersecretion. Th2 cells release IL-4, IL-13, IL-5, and IL-9. When overexpressed in vivo, each Th2 cytokine induces goblet-cell metaplasia in the murine lung. However, only IL-9 directly induces goblet-cell metaplasia and MUC5 gene expression. Thus, it is assumed that the actions of the other Th2 cytokines are via indirect effects on as yet unknown cells or pathways. The effects of IL-9 on MUC5 gene expression and goblet-cell secretion are presumably through its receptor expressed on airway epithelial cells. Likewise, the effects of IL-4 and IL-13 on goblet-cell proliferation are mediated via a receptor complex composed of the IL-4R and the IL-13R1 chains.

	Footnotes

Address correspondence to: Marsha Wills-Karp, Johns Hopkins University, 615 N. Wolfe St., Baltimore, MD 21205. E-mail: mkarp{at}welch.jhu.edu

(Received in original form May 3, 2000).

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