Expression of the Transforming Growth Factor β Isoforms in Inflammatory Cells of Nasal Polyps

ALTHOUGH nasal polyposis is frequently encountered in the practice of otolaryngology, its pathophysiological features are still poorly understood and it remains difficult to cure. Nasal polyposis is a chronic inflammatory disease of the upper airways in which the structural modifications of epithelium (secretory hyperplasia and squamous metaplasia) and lamina propria mucosae (basement membrane thickening, extracellular matrix accumulation, and fibrosis) are frequently associated with inflammatory cell infiltration.¹ Inflammatory cells release various cytokines and growth factors that are likely to regulate other features of nasal polyposis, such as fibroblast proliferation, extracellular matrix production, and epithelial cell proliferation and/or differentiation.² A better understanding of the mechanisms underlying these mucosal alterations and cellular interactions should help physicians design more appropriate therapeutic strategies.

Among the growth factors that are possibly involved in chronic inflammatory diseases of the airways and therefore in nasal polyposis, transforming growth factor β (TGF-β) could play a key role. Transforming growth factor ββ is a 25-kd protein that includes 5 isoforms, but only TGF-β₁, TGF-β₂, and TGF-β₃ have been found in human tissues.^3,4 Transforming growth factor β mediates a broad spectrum of biological activities,^3,4 particularly tissue repair⁵ in most human fibrotic disorders,⁶ and TGF-β is present in the epithelial lining fluid of healthy lower airways⁷ where it can be produced by several cell types, such as macrophages⁸ and epithelial cells.⁹

The aim of this study was to characterize the expression of TGF-β in nasal polyposis. The TGF-β1 gene and TGF-β₁protein were previously thought to be expressed in nasal polyps (NPs), but the expression of TGF-β₂ and TGF-β₃ has not yet been studied in upper airways.¹⁰ In this study, we detected and localized TGF-β and TGF-β isoforms (TGF-β₁, TGF-β₂, and TGF-β₃) in NPs and nasal mucosa using immunohistochemistry and immunoblotting. Moreover, we quantified TGF-β expression and compared it between NPs and nasal mucosa. The expression of TGF-β was also analyzed in relation to morphologic changes of the epithelium. Finally, using double labeling, the expression of TGF-β₁ was evaluated in macrophages and eosinophils.

Twenty-one adult patients with nasal polyposis and 6 healthy controls were included in the study. Patients with cystic fibrosis or primary ciliary dyskinesia were excluded because of the special features of their NPs. The diagnosis of nasal polyposis was established on the basis of medical history and symptoms, endoscopic examination of the nose, and a computed tomographic scan of nasal fossa and paranasal sinuses. Because of the failure of medical treatment (ie, systemic and local corticosteroid therapy for at least 1 year), surgery was found to be necessary to improve the patients' symptoms. Nasal mucosa from NPs were sampled in all patients, and nasal mucosa from the inferior turbinate, free of NPs, was sampled at the same time in 11 patients at the beginning of the surgical procedure (endoscopic endonasal ethmoidectomy). Six adult patients who underwent rhinoplasty were included as controls: all were free of symptoms of nasal inflammation with a normal appearance of the nasal mucosa at endoscopic examination. Nasal mucosa from the inferior turbinate was sampled in controls during the surgical procedure. All patients were asked to stop general and/or local nasal treatment 1 month prior to surgery. This protocol was approved by the review and ethics committee of our institution.

Samples from 17 patients and 6 controls were immediately fixed in formaldehyde, embedded in paraffin, and divided into 5-µm sections. For each sample, a section was systematically stained (hemalum-eosin-safran) for standard histomorphologic analysis. In 4 patients, NPs were sampled, immediately frozen in liquid nitrogen, and then stored at −80°C until examination using Western blot analysis.

Monoclonal mouse immunoglobulin antihuman TGF-β_1-3 (1835-01, Genzyme, Cambridge, Mass ), polyclonal chicken immunoglobulin antihuman TGF-β₁ (BDA-19, R&D Systems, Minneapolis, Minn), polyclonal goat immunoglobulin antiporcine TGF-β₂ (BDA-53, R&D Systems), and polyclonal goat immunoglobulin antichicken TGF-β₃ (BDA-48, R&D Systems) (both goat immunoglobulins cross-reacting with human TGF-β) were used as primary antibodies for immunohistochemistry and Western blot analysis. In addition, monoclonal mouse immunoglobulin anti-CD68 (KP-1, MO814, Dako, Glostrup, Denmark) and monoclonal mouse immunoglobulin antieosinophil cationic protein (ECP) (EG2, 10-9116-01, Pharmacia, Uppsala, Sweden) were used as primary antibodies for immunochemical detection of activated macrophages and eosinophils, respectively.

After deparaffinization, nonspecific antigenic sites were saturated with human AB serum, and the slides were incubated for 30 minutes with primary antibodies diluted at 1:50 except for anti–TGF-β_1-3 (1:10) and anti–TGF-β₂ (1:25). All incubations were performed at room temperature in a moist chamber, and the slides were washed in Tris-buffered saline (0.015 mol/L; pH, 7.6) between each incubation. The detection of TGF-β_1-3 was performed in 17 NP samples, 11 nasal mucosa samples from patients, and 6 nasal mucosa samples from controls, and the detection of TGF-β₁, TGF-β_2, and TGF-β₃ was performed in 10 NP samples with high TGF-β_1-3 expression using biotinylated secondary antibodies and amplification by streptavidin-peroxidase complex (Labeled Streptavidin Biotim kit K680, Dako) with aminoethylcarbazole as the chromogen. In addition, in 4 NP samples with high TGF-β_1-3 expression, simultaneous detection of TGF-β₁ first with CD-68 and second with ECP was also performed (double labeling with alkaline phosphatase and anti–alkaline phosphatase). Transforming growth factor β₁ labeling was revealed using fast red TR salt (Sigma Aldrich Inc, St Louis, Mo), and then CD-68 or ECP labeling was revealed using fast blue BB salt (Sigma Aldrich Inc). Tissue sections were finally counterstained with hematoxylin except for those with double labeling when green-light counterstaining was performed. The primary antibody was omitted on 1 tissue section in all labeling experiments to serve as a negative control.

All tissue sections were examined under blind conditions with a light microscope by 2 independent observers. The predominant morphologic type of the epithelium was recorded as pseudostratified ciliated epithelium, secretory hyperplasia, or squamous metaplasia. In addition, the presence of inflammatory cells in the lamina propria was evaluated in at least 10 randomly selected fields (final magnification ×500) and scored in each field as follows: −, when inflammatory cells were absent; +, when less than 10 cells were present; ++, when more than 10 but less than 30 cells were present; and +++, when more than 30 cells were present. The result was expressed as the mean score for each sample. The analysis of TGF-β_1-3 labeling included the quantification of TGF-β expression, which was achieved by counting the total number of cells positive for TGF-β_1-3 located in the lamina propria underlying the surface epithelium in 10 randomly selected fields (final magnification ×500). For each sample, a TGF-β index was therefore established as an absolute number. In addition, we recorded the predominant morphologic type(s) of epithelium (see earlier description) overlying the areas of the lamina propria with marked TGF-β_1-3 positivity and assessed the presence of TGF-β_1-3–labeled cells in surface epithelium. The proportion of double-labeled cells among cells positive for CD-68 or ECP was estimated by TGF-β₁ double labeling.

Transforming growth factor ββ was extracted from frozen NP samples using a previously published method.¹¹ Nasal polyp extracts (80 µg of proteins on each lane) were run under nonreducing conditions on a 15% sodium dodecyl sulfate polyacrylamide gel and transferred overnight in a Tris glycine buffer (pH, 8.3) with 20% methanol onto a nitrocellulose membrane (0.45-µm pore size, Schleicher & Schuell, Keene, NH). Five nanograms of purified TGF-β₁ (101-B1, R&D Systems), TGF-β₂ (101-B2, R&D Systems), or recombinant TGF-β₃ (243-B3, R&D Systems) was run on 1 lane of each blot to act as a positive control. Immediately following transfer, the proteins were cross-linked onto nitrocellulose membranes by dipping them in 0.5% glutaraldehyde phosphate–buffered solution. After blocking with 1% nonfat dry milk in Tris buffer (pH, 7.5), the membranes were then incubated overnight at 4°C, with anti–TGF-β_1-3 diluted to 1:1000 and the various anti–TGF-β isoform antibodies diluted to 1:400 in 1% milk Tris polysorbate buffer. After rinsing, membranes were incubated with secondary biotinylated antibodies diluted to 1:4000 for 1 hour. The membranes were then incubated with streptavidin-peroxidase complex (RPN 1051, Amersham, Uppsala) for 45 minutes and revealed using the electrochemoluminescence detection method (RPN 2109, Amersham).

The nonparametric Wilcoxon test was used to compare TGF-β_1-3 indices between NP and nasal mucosa from patients. The nonparametric Mann-Whitney U test was used to compare TGF-β_1-3 indices between NP or nasal mucosa from patients and nasal mucosa from controls. All analyses were performed using a personal computer (Apple, Cupertino, Calif) with Instat 2.00 software (Instat, Graphpad, San Diego, Calif).

Modifications of epithelial morphologic features were frequent in NP samples (predominance of pseudostratified ciliated epithelium in 11 cases, squamous metaplasia in 3 cases, and secretory hyperplasia in 4 cases), but exceptional in nasal mucosa from patients (1 case with predominance of squamous metaplasia) and nasal mucosa from controls. As expected, inflammatory cells were more numerous in NP samples than in nasal mucosa from patients, but were rare in nasal mucosa from controls (data not shown).

Using immunohistochemistry, TGF-β_1-3 cell labeling was always detected in NP samples (17/17), sometimes in nasal mucosa samples from patients (5/11), and seldom in nasal mucosa from controls (1/6). In lamina propria and epithelium, the labeling was restricted to scattered cells confined to either the plasma membrane or the cytoplasm (Figure 1) without imumunoreactivity in the extracellular matrix. In the lamina propria, the TGF-β_1-3 indices were significantly higher in NP samples (mean ± SD, 55 ± 17.4 arbitrary units) than in nasal mucosa from controls (mean ± SD, 0.2 ± 0.4 arbitrary units; P<.001) and nasal mucosa from patients (mean ± SD, 10.5 ± 20.6 arbitrary units; P<.05) (Figure 2). The difference between TGF-β_1-3 indices in nasal mucosa from patients and nasal mucosa from controls was not statistically significant (P>.05). In epithelium, TGF-β_1-3–positive cells were detected more frequently in NP samples (7/17) than in nasal mucosa from patients (2 of 11) and nasal mucosa from controls (1 of 6), but only in small numbers. The morphologic characteristic of the epithelium overlying the areas of the lamina propria with intense TGF-β_1-3 positivity was either normal or showed secretory hyperplasia (Figure 1), but never showed squamous metaplasia.

In Western blot analysis using anti–TGF-β_1-3 antibody, a strong TGF-β band of 25 kd was observed in NP protein extracts (Figure 3).

Using immunohistochemistry in 10 NP samples, TGF-β₁ was always detected in numerous cells of the lamina propria (Figure 1), while TGF-β₂ and TGF-β₃ were rarely detected in the same NP sample (data not shown).

In Western blot analysis, a strong TGF-β₁ band of 25 kd was detected in NP samples, but only a weak TGF-β₂ band and no TGF-β₃ bands were detected (Figure 3).

Using double immunolabeling, we detected TGF-β₁ with CD-68 and ECP in numerous cells from the lamina propria in the 4 studied NP samples (Figure 4). A semiquantitative evaluation showed that even if ECP-positive cells seemed to be more abundant than CD-68–positive cells in the 4 NP samples, a larger proportion of CD-68–positive cells than ECP-positive cells was also positive for TGF-β₁.

The major roles played by TGF-β in chronic inflammatory reactions, extracellular matrix accumulation, and epithelial growth and differentiation have been recently emphasized in many cell culture and animal models.^12-14 Although recent studies have dealt with TGF-β expression in human lung diseases and especially fibrotic diseases, it has not yet been precisely characterized in human upper airways. In this study, we evaluated TGF-β expression in nasal polyposis, in which many histomorphologic modifications are observed (eg, epithelial changes, basement membrane thickening, and inflammatory cell infiltration with eosinophils).¹ Our results obtained from nasal tissues using either immunohistochemistry or Western blot analysis were strictly concordant, showing that TGF-β was strongly expressed in NP samples almost exclusively in the β₁ isoform.

As previously reported, we detected TGF-β only in NPs and inflammatory mucosa but not in nasal mucosa from controls.¹⁰ Transforming growth factor ββ was mainly detected in the lamina propria, where it was localized in numerous isolated cells exhibiting inflammatory cell morphologic features. This finding suggests that in the upper airways, as in other tissues, the main source of TGF-β could be the inflammatory cells. In this study, we evaluated the cell expression of TGF-β₁ in eosinophils in NPs, which have been previously suspected to express the TGF-β1 gene in NPs¹⁰ and macrophages that are known to be able to produce TGF-β.⁸ Transforming growth factor β₁ was present in many cells of each type, but not in all. Although eosinophils seemed to be more numerous than macrophages in the NPs, TGF-β₁ was detected more frequently in macrophages than in eosinophils, contrasting with findings of a previous study.¹⁰ This discrepancy could result from technical differences concerning the method of detection of eosinophils and macrophages, but also from the level at which TGF-β was detected (protein vs mRNA) in these cells. Apart from the lamina propria, TGF-β was also detected in some epithelial areas, suggesting local production, since airway epithelial cells are able to synthesize TGF-β in vitro⁹ and are an important site of TGF-β expression in advanced pulmonary fibrosis.¹⁵ In any case, TGF-β expression in epithelial cells was always low compared with its expression in inflammatory cells. A weak immunoreactivity of the extracellular matrix was detected with the TGF-β isoform–specific antibodies but not with the TGF-β_1-3–specific antibody (Figure 1). This discrepancy could be interpreted as a technical difference between the polyclonal and monoclonal nature of the antibodies. Another interpretation could be that the anti–TGF-β_1-3 monoclonal antibody detects a secreted form of TGF-β, while TGF-β isoform–specific antibodies detect both secreted and matrix-associated TGF-β forms.

Characterization of TGF-β expression by immunohistochemistry and Western blot analysis showed that TGF-β₁ was the main isoform of the TGF-β family expressed in NPs. Conversely, TGF-β₂ and TGF-β₃ were expressed at a low level in NPs. These findings contrast with TGF-β expression in distal lower airways where the 3 TGF-β isoforms were detected.¹⁵

The major expression of TGF-β in NPs suggests that TGF-β, particularly TGF-β₁, could be involved in the pathogenesis of nasal polyposis. The TGF-β present in NPs could participate in the complex regulation of local inflammation. Transforming growth factor β₁ is a potent chemoattractant for neutrophils and mononuclear cells,¹⁶ but also has immunosuppressive effects on T lymphocytes,¹⁷ neutrophils,¹⁸ and macrophages.¹⁹ Further studies could help define how TGF-β can modulate local inflammation in nasal polyposis. Transforming growth factor ββ is a chemoattractant for fibroblasts,²⁰ greatly enhancing fibroblast synthesis of extracellular matrix proteins on one hand⁶ and down-regulating the expression of matrix degradation enzymes on the other.³ Transforming growth factor ββ could therefore be involved in the pathogenesis of nasal polyposis by enhancing extracellular matrix accumulation, which seems an important mechanism of NP growth as suggested in experimental models.^21,22 Another important effect of TGF-β is its ability to influence airway epithelial cell growth and differentiation. We²³ previously showed that epithelial cell proliferation was increased in NPs and that this epithelial cell proliferation could be induced by mitogenic growth factors that are locally produced by inflammatory cells.²⁴ Transforming growth factor β₁, which has been shown in vitro to down-regulate epithelial cell proliferation,^9,13,25 could limit the proliferative effects of other growth factors to control the epithelial cell proliferation in NPs. Transforming growth factor β₁ and TGF-β₂ have been reported to induce in vitro squamous metaplasia in airway epithelial cells,^9,13 but we never detected TGF-β–positive cells near areas of epithelial squamous metaplasia. This result is consistent with a recent in vitro study in which TGF-β failed to induce squamous differentiation in human bronchial epithelial cells.²⁶ In contrast, we observed that secretory hyperplasia was frequently associated with high local TGF-β expression. This finding suggests that TGF-β could induce mucus cell differentiation in upper airways, as previously proposed.²⁷

In the present study, we showed that TGF-β was present in NPs and to a lesser extent in chronic inflammatory mucosa, but not in nasal mucosa from upper airways in controls, suggesting that TGF-β could be involved in the pathophysiological development of nasal polyposis. Of the various TGF-β isoforms, only TGF-β₁ was significantly detected. Transforming growth factor β₁–labeled cells were mainly macrophages and eosinophils, implying that these inflammatory cells could be a major local source of TGF-β. A broad and complex spectrum of effects can result from TGF-β release in the upper airways, such as epithelium changes and extracellular matrix accumulation. As therapeutic strategies with anti–TGF-β molecules are currently being developed for other inflammatory diseases,⁶ a better understanding of TGF-β activity in nasal polyposis could have direct clinical implications in the future.

Accepted for publication March 13, 1998.

This work was supported by grant CRC96059 from the Délégation à la Recherche Clinique de L'Assistance Publique des Hôpitaux de Paris, Paris, France. We thank the staff from the operating theater suite and photography laboratory of the Hôpital Intercommunal, Créteil, France, for their contribution to the collection of samples and photomicrographs.

Corresponding author: André Coste, MD, Service d'ORL et de Chirurgie Cervico-Faciale, Centre Hospitalo-Universitaire Henri Mondor, 51 Avenue du Maréchal de Lattre de Tassigny, 94010 Créteil Cédex, France (e-mail: coste@im3.inserm.fr).