As the previous section highlighted, there are two interesting similarities between NP and asthma. First of all there is eosinophil-dominated inflammation. Secondly, there are structural modulations of the nasal mucosa, with the myofibroblast being thought to play an important role. This section addresses these two features. We will also explain the central coordinating role of the epithelium in these two processes. A better understanding of these essential interactions can help us to understand the pathogenesis of NP better.
Eosinophilic inflammation: a ‘toxic’ source of mediators
The polyp tissue is infiltrated predominately by eosinophils, lymphocytes, plasma cells and mast cells (16–19).
The activated infiltrating eosinophils produce a large amount of toxic proteins, such as eosinophilic cationic protein (ECP) and major basic protein (MBP). In addition to these toxic mediators, eosinophils are also capable of producing a variety of cytokines, chemokines and growth factors. For instance, they produce interleukin-5 (IL-5), GM-CSF, RANTES and GRO-α (15, 17, 20–23). So they seem to extend their own lifespan and increase tissue infiltration in an autocrine fashion.
It is, however, not known what initiates the influx of activated eosinophils into the nasal polyp. It is widely accepted that eosinophils are also a hallmark of allergy. In patients with NP and co-existing allergic rhinitis the eosinophils seem to be attracted mainly by the release of IL-5 (15). Preliminary data in nonallergic, nonasthmatic and aspirin-tolerant NP patients however do not show increased levels of IL-5-producing cells (unpublished results). By contrast, in the absence of allergy, eosinophils appear to be recruited mainly by the release of GM-CSF (15). Nevertheless, the resulting eosinophilic influx appears to be the same for both atopic and nonatopic NP (24–27). This does not hold true for asthmatic and aspirin-intolerant patients with NP. Bachert et al. found significantly more eosinophilic infiltration in NP samples containing high total IgE tissue concentrations (10). These high total IgE levels were more frequently found in asthmatic and aspirin-intolerant NP patients. Other researchers have confirmed that the eosinophilic influx is higher in asthmatic patients when compared with nonasthmatic patients (10–14). This difference in eosinophilic influx is even more marked in aspirin-intolerant asthmatic patients (26, 28–30). This suggests a more aggressive inflammatory response in those patients who apparently have more extensive polyposis and many recurrences after surgery. This correlation between the extent of eosinophilia and the disease severity is also seen in asthma itself. Patients with a higher degree of eosinophilia have significantly more severe symptom scores and residual airway obstruction after bronchodilatory therapy (31–34).
Given that nasal polyps are formed in a defined region in the nose it is important to note that there seems to be a specific distribution of eosinophils in the nasal cavity. As can perhaps be expected, there are significantly more activated eosinophils in polyp samples compared with middle and inferior turbinates from healthy controls (16, 35, 36). However, several authors have specifically investigated the distribution of eosinophils throughout the nose in NP patients. They report that polyp samples contain a significantly higher amount of eosinophils than the middle and inferior turbinate samples in the same NP patients, with a significantly higher number of eosinophils in the middle turbinate than in the inferior turbinate (16, 36, 37).
In conclusion, we can state that it is not known what initiates the primary recruitment of eosinophils to the site of nasal polyps. In allergic rhinitis there is a role for Th2-lymphocytes producing IL-5 and this may apply to NP. To address this question the cellular sources of Il-5 need to be identified.
Structural modulations of the nasal mucosa: the role of the myofibroblast
Myofibroblasts are atypical stromal cells that play a crucial role in the pathological tissue changes seen in both NP and asthma. (Electron) microscopic examinations of nasal polyps reveal a number of typical findings. First, the surface area of the polyp can vary considerably. The polyp surface is partially covered by normal (ciliated) respiratory epithelium, interrupted by areas of erosion/damage and squamous metaplasia. Typically, there is goblet cell hyperplasia. Secondly, underneath this partially damaged epithelium, there is thickening of the basal membrane (lamina reticularis). And thirdly, the stroma of the polyps is characterized by massive oedema, pseudocysts, and (subepithelial) fibrosis with an accumulation of extracellular matrix (38–43).
Interestingly, these observations are very similar to the observations in the sinus mucosa of CRS patients without NP (13, 27), and to observations in the lower airways of asthmatic patients (44–49). These typical findings are more pronounced in the nasal mucosa of CRS/NP patients with concomitant asthma (13).
Both in asthma and NP, pathological cells have been observed underneath the thickened basal membrane that are not present in ‘healthy’ bronchial or nasal mucosa. (12, 50–53). They are myofibroblasts, which can be seen as an activated phenotype of fibroblasts. Upon stimulation with transforming growth factor-β (TGF-β), resident fibroblasts in skin, cardiac, lung and nasal tissue differentiate into active myofibroblasts where, in nonpathological circumstances they are involved in wound repair and tissue differentiation (54–58).
Myofibroblasts produce large amounts of extracellular matrix molecules, such as collagens (type I, III, IV and VIII) and fibronectin. This extracellular matrix secretory function is essential in tissue repair and wound healing. The cytoplasm of the myofibroblast contains a fibronexus that connects the stress fibres of α-smooth muscle actin, through a trans-membrane αβ integrin, to the fibronectin in the extracellular matrix. Contraction of the myofibroblasts pulls the extracellular matrix together and reduces the physical size of a damaged area. The tissue repair process is completed by the apoptosis of the myofibroblast.
Myofibroblasts play an important role in several diseases. One of those is asthma, in which an interaction between the epithelium and myofibroblasts is at the basis of the underlying disease mechanism. We hypothesize that similar interactions are of importance in the pathogenesis of NP. The epithelium is a crucial factor in these interactions, as will be clarified in the following section.
Epithelium: active participant in the inflammatory and structural response
Traditionally, the nasal epithelium is seen as a passive barrier lining the nasal cavity, protecting the tissue against all sorts of pathogens and allergens. However, there is a growing awareness that it should rather be seen as an active participant in the immunological response. In NP, the epithelium is both an active player and a ‘passive’ target in the pathology. It plays a central role in the interactions with eosinophils and myofibroblasts, as will be explained below.
The epithelium is ‘passively’ under attack by the infiltrating eosinophils. The activated eosinophils produce reactive oxygen radicals, as well as toxic proteins (ECP and MBP) that have been shown to damage respiratory epithelium (59, 60). Ciliary beat frequency, as well as epithelial cell membrane integrity, is significantly decreased by activated eosinophils (61–67). Furthermore, cell proliferation is significantly higher in the polyp epithelium compared with inferior turbinate epithelium from the same patients (68). Epithelial damage caused by inflammatory mediators induces this proliferation via epithelial repair processes.
It seems that the epithelium itself is partly responsible for the toxic eosinophilic invasion. In a model where epithelial damage is mimicked by nonenzymatic induction of loss of cell–cell contacts, a pleiotropic induction of mediators is seen. These mediators are known to be involved in repair and fibrosis (IL-1β, TGF-β, epidermal growth factor [EGF], vascular endothelial growth factor [VEGF], insulin-like growth factor-binding protein 3 [IGF-BP3], TIMP-1 and TIMP-2), but they are also involved in the recruitment and activation of cells of the immune system (IL-6, IL-8, G-CSF, IL1-β, γ-interferon, tumour necrosis factor-α, IL-4, IP-10, leukemia inhibitory factor (LIF) and GRO-α) (A.B. Vroling, personal communication,). Several researchers have shown that polyp epithelial cells additionally produce a number of eosinophil chemo-attractant mediators. These include GM-CSF, RANTES and eotaxin (15, 69–73).
Not only can epithelial cells attract eosinophils to the polyps, they can also increase their lifespan. When incubating peripheral blood eosinophils from healthy subjects with human nasal polyp epithelium cell conditioned medium (HECM), eosinophil survival increased significantly as a result of the inhibition of apoptosis. It was possible to block this effect almost completely through the addition of anti-GM-CSF to the HECM (69, 70, 72). This indicates that GM-CSF is the most important eosinophil survival enhancer produced by the nasal polyp epithelial cells.
The epithelium also plays a very active role in the interaction with myofibroblasts. Under normal circumstances this interaction is essential to organogenesis. For instance, epithelial–myofibroblast interactions are critical in embryologic lung development. They secrete soluble mediators, growth factors and interstitial matrix and/ or basement membrane molecules. In this process, platelet-derived growth factor (PDGF) is essential for the myofibroblast proliferation. Bostrom et al. demonstrated that postnatal surviving PDGF-A-deficient mice develop pulmonary emphysema secondary to the failure of alveolar septation. This is caused by the loss of alveolar myofibroblasts and associated elastin fibre deposits (74). Given that TGF-β is important for myofibroblast differentiation it should be noted that it also induces PDGF receptors on the myofibroblast. Other mediators considered to be important in myofibroblast activation and proliferation are TGF-α, EGF, GM-CSF, FGF and IGF (75).
Myofibroblasts are frequently present in asthma, where a specific interaction between epithelium and these fibroblasts is thought to be part of the underlying disease mechanism. As mentioned before, asthma (that often co-exists with NP) shares many similar histopathological features with CRS and/or NP. There is epithelial damage, thickening of the lamina reticularis, hyperplasia of goblet cells and smooth muscle cells, accumulation of extracellular matrix, and fibrosis. This structural reorganization in the bronchial mucosa of asthmatic patients is known as ‘airway remodelling’ and is responsible for the irreversibility of the disease. An essential component of this pathological remodelling process is the interaction between epithelial cells and myofibroblasts. This may take place through a reactivation of the ‘epithelium mesenchymal trophic unit’ that plays an important role in normal lung development (46, 49). As in the process of deranged wound repair and tissue fibrosis, it is not clear what triggers this revival of a system that is essential to normal organ development. The higher prevalence of NP in asthmatic patients is, however, a striking finding that forces us to consider the possibility that similar pathology might underlie both diseases.
Myofibroblasts are found in pathological conditions that may have some bearing on the presence of these cells in NP. First, under conditions of unchecked, deranged or repeated tissue repair, the myofibroblasts do not go into apoptosis. This could lead to fibrosis (75). In NP, the highest density of myofibroblasts is found in the pedicle area of the polyp. This area, where the polyps can be thought to ‘grow’, also contains the highest density of TGF-β-positive cells (51). High levels of TGF-β are secreted by the infiltrating eosinophils, and very likely by the epithelium also (76, 77). The polyp epithelium might be blocked in a ‘repair phenotype’, with the continuous release of proliferative and profibrotic mediators. Zhang demonstrated this in vitro. He chemically damaged cultured bronchial epithelial cells with poly-l-arginine, which is a surrogate for ECP. In a co-culture system these damaged epithelial cells significantly increased myofibroblast differentiation through the release of high amounts of TGF-β, bFGF, IGF-1 and PDGF (78). When mechanically damaging cultured guinea-pig bronchial epithelial cells, Morishima et al. saw a similar increase in myofibroblast proliferation, accompanied by the up-regulation of collagen type I and III synthesis by co-cultured (myo-) fibroblasts (79). In this way, fibrotic disease can be seen as a major pathological end point of activated and proliferating myofibroblasts. Indeed, myofibroblasts have been identified as major players in fibrotic diseases such as: pancreatic fibrosis, liver fibrosis and cirrhosis, sclerosing glomerulonephritis, fibrosis in Crohn's disease, pulmonary fibrosis and many others. In NP however, fibrotic changes do not dominate. Fibrosis is only evident in the peduncle area, where most myofibroblasts are found. Most of the polyp stroma, however, is predominantly oedematous. Nevertheless, the presence of myofibroblasts in NP points to a pathological damage-repair response in which the created molecular environment allows them, together with the activated eosinophils, to escape normal apoptosis. But what derails this ‘normal’ repair process? What exactly triggers the epithelium to persist in this ‘activated’ phenotype and to inhibit myofibroblast and eosinophil apoptosis?
Unfortunately, we do not know the answers to the above formulated questions. As will be discussed in the section Concluding remarks and future directions, we intend to design several experiments that may enhance our understanding of the complex interaction of epithelium and myofibroblasts in NP. We expect to gain more insight into the pathophysiology of NP, with the aim of identifying potential new targets for treatment.