Review article: Nasal polyposis: a cellular‐based approach to answering questions

Nasal polyposis (NP) is a common chronic inflammatory disease of the nasal mucosa that has a major impact on patients' lives. NP is characterized by benign polypous tissue swellings in the nose that originate from the paranasal sinuses, most often from the anterior ethmoid complex (1, 2) (1). From there the polyps can descend between the middle turbinate and the lateral nasal wall into the nasal cavity causing symptoms such as nasal congestion, rhinorrhea, hyposmia and facial pressure (2). Nasal endoscopic view of the left nasal cavity. Nasal polyps are transparent, pale grey oedematous projections originating from the nasal ethmoid mucosa, descending between the middle turbinate and the lateral nasal wall into the nasal cavity. This polyp just touches the superior border of the inferior turbinate. Coronal reconstruction of computed tomography (CT) scanning image of nasal polyposis in a 48-year-old Turkish man. There is opacification of all sinuses and complete obstruction of both nasal cavities. Treatment with corticosteroids alleviates symptoms, but no curative treatment exists. Often patients require recurrent operations and this, in combination with the symptoms, has a significant effect on the patients' quality of life (3, 4). When tested by means of a disease-independent questionnaire (SF-36), the quality of life in these patients is worse than in patients suffering from hypertension, migraine, angina pectoris and head and neck cancer. NP patients have comparable quality of life scores as patients with chronic obstructive pulmonary disease (5). Unfortunately, the aetiology of NP is largely unknown. Although some hypotheses have focused on the possible involvement of micro-organisms in the aetiology of NP, this has not yet developed into a successful treatment alternative. This review aims at discussing some of the difficulties and pitfalls in NP research, and to identify the important cellular players and interactions in the pathophysiology of NP. We would also like to suggest potential relevant future directions for research. Understanding the pathogenesis of NP may lead to new treatment options for this incapacitating disease. Fundamental research into the pathogenesis of NP is hampered by two problems. First, it is unclear how the many different clinical phenotypes of NP influence the pathogenesis. Secondly, it is not clear whether NP should be considered a local disease or a local manifestation of a systemic disease. Many co-morbidities have been described in NP that affect the prevalence of NP. In the general population the prevalence is 0.5–4.3%, making it one of the most common chronic diseases of the upper respiratory tract. The prevalence of NP is increased in patients with asthma (7–15%), cystic fibrosis (39–56%) or aspirin intolerance (36–96%). Interestingly, although the prevalence is increased in asthma, this does not seem to hold true for patients with allergic rhinitis, where the prevalence of NP is unchanged (0.5–4.5%) (2, 6). Chronic rhinosinusitis (CRS) almost always coexists with NP, but the converse is not true, only about 20% of the patients with CRS develop nasal polyps (7). Evidence accumulates that CRS with NP and CRS without NP actually are two different disease entities (8, 9). As these different clinical NP phenotypes have a clear influence on the prevalence of NP, they cannot be discarded. We do not know whether features seen in NP are a local manifestation of these co-morbidities. For instance, similar typical findings can be found in microscopic examinations of nasal polyps, when compared with the bronchial mucosa of patients with asthma. In both tissues there is epithelial damage, goblet cell hyperplasia, thickening of the basement membrane, accumulation of extracellular matrix, fibrosis and eosinophil-dominated inflammation. The link between these two diseases is further made plausible by the observation that the nasal polyp eosinophilic inflammation is significantly higher in NP patients with concomitant asthma when compared with nonasthmatic NP patients (10–14). Another issue in these NP phenotypes is that different possible mechanisms can lead to the same polyp formation. For instance, as will be seen in the section Eosinophilic inflammation: a 'toxic' source of mediators, there appears to be a difference in eosinophil recruitment between allergic (IL-5) and nonallergic nasal polyps [granulocyte macrophage-colony stimulating factor (GM-CSF)] (15). Yet the amount of tissue eosinophilia seems to be the same in both groups. These examples indicate that NP is a heterogeneous disease. In order to get to the bottom of the NP mystery we must study the differences as well as the similarities between the different co-morbidities. We have to be very cautious when interpreting data from experiments, where NP phenotypes are combined in one group and compared with healthy nasal mucosa. It is very difficult, if not impossible, to determine which factors can be attributed to the heterogeneity of the underlying diseases and which factors are essential in the development of NP. Designing future experiments to seek actively for differences as well as similarities between the clinical phenotypes of NP will shed more light on the pathogenesis of NP. A similar discussion is possible on the topic of local versus systemic disease. As will also be seen in the section Eosinophilic inflammation: a 'toxic' source of mediators, the inflammatory process (eosinophils, lymphocytes, etc.) varies significantly in polyps, the middle turbinate and the inferior turbinate in the same patient. This may suggest that NP is a local disease, or that the inflammatory process in the middle turbinate is (i) different from that in the inferior turbinate and (ii) probably closer to the process in NP. This is an important finding, as much NP research has focused solely on inferior turbinate samples from healthy patients as control tissue for NP. We have to make sure that the 'healthy' control tissue used is from the exact same source as the diseased nasal polyp. As nasal polyps mostly originate in the ethmoid sinuses near the middle turbinate, this would either be healthy sinus or middle turbinate mucosa. Future experiments should be designed to identify the best control tissue. 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. 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. 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. 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 by the nasal polyp epithelial The epithelium also plays a very active role in the interaction with normal circumstances this interaction is essential to For instance, interactions are in lung They mediators, growth factors and matrix or basement membrane In this growth factor is essential for the myofibroblast et al. that develop pulmonary to the of This is caused by the loss of myofibroblasts and Given that is important for myofibroblast differentiation it should be that it also induces on the myofibroblast. Other mediators considered to be important in myofibroblast activation and proliferation are GM-CSF, and Myofibroblasts are frequently present in asthma, where a specific interaction between epithelium and these fibroblasts is thought to be of the underlying disease mechanism. As asthma often with many similar features with CRS NP. There is epithelial damage, thickening of the of goblet cells and muscle accumulation of extracellular matrix, and This structural in the bronchial mucosa of asthmatic patients is known as and is responsible for the of the disease. essential of this pathological process is the interaction between epithelial cells and This may through a of the that plays an important role in normal lung development As in the process of wound repair and tissue it is not clear what this of a system that is essential to normal The higher prevalence of NP in asthmatic patients is, however, a that us to the that similar both diseases. Myofibroblasts are found in pathological that may have some on the of these cells in NP. First, under of or tissue the myofibroblasts do not into apoptosis. This lead to fibrosis In NP, the of myofibroblasts is found in the area of the polyp. This where the polyps can be thought to also contains the of cells levels of are by the infiltrating eosinophils, and very by the epithelium also The polyp epithelium be in a with the release of and mediators. this in damaged bronchial epithelial cells with which is a for In a system these damaged epithelial cells significantly increased myofibroblast differentiation through the release of high amounts of TGF-β, and When bronchial epithelial et al. a similar increase in myofibroblast by the of and by fibroblasts In this disease can be seen as a major pathological of activated and myofibroblasts have been as major players in diseases such fibrosis and fibrosis in disease, pulmonary fibrosis and many In NP however, changes do not is only in the where most myofibroblasts are of the polyp however, is Nevertheless, the of myofibroblasts in NP to a pathological response in which the together with the activated eosinophils, to normal apoptosis. what this repair the epithelium to in this phenotype and to myofibroblast and eosinophil Unfortunately, we do not know the to the As will be in the section and future we to several experiments that may understanding of the complex interaction of epithelium and myofibroblasts in NP. We to more into the pathophysiology of NP, with the of potential new for The for new treatment is there is no curative treatment for NP. This section will on the present treatment options in NP. We will the the as well as on we will a investigated of a in order to explain how research can lead to new In an extensive review of the on treatment in NP, the on and Nasal an treatment for NP (2). are the for NP. This can from or in to to a of systemic in patients. There is a amount of that as well as systemic are in the size and symptoms of nasal polyps Nevertheless, a number of patients appear to be to or seem to develop a the So do some patients to treatment do One important factor is whether the local of the polyp. 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This IgE appears to be that can as that can large of (10). is also one of the in patients with atopic In patients with atopic the of has been shown to although This potential effect of has not yet been in NP, but are being have the possible involvement of in the pathogenesis of NP. these have been mostly were found in both healthy and NP patients. no treatment has been considered or is being In NP, eosinophils are the infiltrating inflammatory to this toxic is a In asthma, this has been by means of the of This in a in blood eosinophils and in a of of airway eosinophils There was also a significant in the number of airway eosinophils, and in the in bronchial Nevertheless, treatment not result in a in It was that but not airway eosinophils. This suggests that IL-5 is either not the most or not the factor responsible for eosinophil infiltration and survival in the asthmatic bronchial mucosa. et al. tested the of in the treatment of NP In a nasal polyp was by means of nasal and compared and after together with symptom scores and nasal There were no significant changes in the symptom scores or in the nasal between the treatment compared with the like in the asthma these researchers observed a complete in blood but only a in tissue interesting observations from on the endoscopic in the nasal polyp after the patients who the treatment were into and the were shown to have significantly higher IL-5 concentrations in the nasal than et al. that IL-5 is not the only responsible for eosinophil In the absence of allergy, eosinophils in nasal polyps seem to be recruited mainly by the release of GM-CSF rather than IL-5 (15). these observations suggest that the treatment effect of perhaps be to those patients atopic means they have increased levels of Treatment would be to the in eosinophilic Unfortunately, the atopic of the patients in the has not been a amount of research, the responsible for the of polyps need to be and many (i) As NP is frequently seen with is NP of the or are these two (ii) As polyps are found to originate in such a defined should we NP as a local disease, or are the polyps a local of a systemic In what do the co-morbidities affect the of nasal This review important cellular players in the pathogenesis of NP on that to identify potential new for To discussion it would seem clear that there is no responsible for NP. To it more even that there is a complex cells inflammatory cells and cells The interaction between these players is not one from eosinophils can damage epithelium, yet factors from the epithelium attract and eosinophils. interactions can be seen between epithelium and These interactions seem to a a with a of the disease. A better understanding of these complex interactions may help us to this and new treatment on understanding of the role of eosinophils in disease and the control of their differentiation and the treatment with was seen as a Unfortunately, this has only been partly as not all patients have from suggests that only patients with high levels of IL-5 have NP is a heterogeneous and very complex disease, in which IL-5 is not the only relevant for eosinophils. As this review IL-5 may even be in nonallergic NP. research has focused on a number of as for a of The development of however, us to for to protein mediators that even in circumstances where amounts of are a more can be A similar can be by the of where of can be it be that the of the treatment is on as seen in or This is but it may well be that the treatment with a of NP. In patients with asthma, the prevalence of polyposis is not only but these patients also have more severe of the it is clear that in the of NP one should not only they may but also they may shed light on the pathogenesis of the disease. The data suggest that this same may hold true for the of control tissue may well be different on the in the nose where they are The cellular influx is different in the inferior and middle and may be different from the influx seen in nasal polyps. a better understanding of differences in the nose it is not clear whether inferior turbinate from healthy are the most The of the treatment should not the of many IL-5 would have been considered as a treatment However, it also that a more understanding of NP is Given the many between seen in asthma and is for the interaction between the polyp epithelium and This interaction may explain epithelial damage and the recruitment and survival of inflammatory differentiation of myofibroblasts, of extracellular matrix, A and seems best to identify the different players involved in this that the pathogenesis of NP. has not been used frequently in NP research. et al. investigated the difference in between nasal mucosa from patients with allergic rhinitis with and without NP They found that were significantly different between the two groups. Several of these for inflammatory mediators, but the with the were described in with of such as were found by et who compared the of NP samples with normal sinus mucosa They also found of several as well as involved in apoptosis Unfortunately, the NP patients were made of a heterogeneous population of patients, with some concomitant asthma, or aspirin Furthermore, the samples used for cell with their typical in cellular influx between make it to from the we to a better of the relevant interactions to we to a at the NP epithelium, which seems to be such a central Not only as a potential target for toxic or factors that may disease, but also of potential role in the of disease through interaction with myofibroblasts and on a cell in combination with can make the a very and that may help us to of the patients in this from co-morbidities such as allergy, asthma, aspirin intolerance or cystic We have defined groups. The group contains healthy subjects from will be from the middle turbinate as well as the The group of patients with without NP middle turbinate from these patients will be The group of patients with CRS and NP In addition to the NP we will also from the middle The epithelial cells will be and Epithelial cell is compared between the different tissue samples and This us to several relevant First, what factors by the epithelium may to the pathogenesis of NP and will be found in polyps with healthy sinus NP middle turbinate with healthy middle turbinate and CRS middle turbinate with healthy middle turbinate Secondly, is NP a local disease or a systemic the polyp samples with the middle turbinate from NP patients will shed more light on this can we the middle turbinate as control tissue for a In is there a significant difference in between the sinus and middle turbinate mucosa in a healthy is the disease in a with CRS and polyposis different from CRS patients without the middle turbinate from NP patients with the middle turbinate from CRS patients will this interesting question In a we will to the question of whether NP is a disease by the co-morbidities or whether nasal polyps are a common found in different disease the differences between the of NP asthma, aspirin intolerance or cystic will us to this Furthermore, by the similarities between these we may further the observations of and common factors. Epithelial cell is compared between middle turbinate nasal polyp (NP) and sinus mucosa in the different and most potential new will be investigated in more in an in cell system we have developed to study their to the interaction between epithelium, fibroblasts and inflammatory