Regional differences in the expression of innate host defense molecules in sinonasal mucosa
2013; Elsevier BV; Volume: 132; Issue: 5 Linguagem: Inglês
10.1016/j.jaci.2013.05.042
ISSN1097-6825
AutoresSudarshan Seshadri, M. Rosati, David C. Lin, Roderick G. Carter, James E. Norton, Andrew W. Choi, Lydia Suh, Atsushi Kato, Rakesh K. Chandra, Kathleen E. Harris, Hong Wei Chu, Anju T. Peters, Bruce K. Tan, David B. Conley, Leslie C. Grammer, Robert C. Kern, Robert P. Schleimer,
Tópico(s)Immune Response and Inflammation
ResumoNasal mucosal epithelial cells form the first line of defense by maintaining a barrier to restrict potentially harmful airborne substances and pathogens. To aid in host defense, specialized epithelial cells in submucosal glands as well as on the mucosal surface secrete mucous that helps to immobilize pathogens and other harmful substances. Beneath this mucous blanket resides an aqueous surface liquid layer that allows proper ciliary function to clear the mucous and entrapped pathogens. In concert with the maintenance of a physical barrier, airway epithelium secretes dozens of antimicrobial peptides and proteins that become incorporated into the mucous and aqueous lining fluids.1Kato A. Schleimer R.P. Beyond inflammation: airway epithelial cells are at the interface of innate and adaptive immunity.Curr Opin Immunol. 2007; 19: 711-720Crossref PubMed Scopus (261) Google Scholar, 2Ooi E.H. Wormald P.J. Tan L.W. Innate immunity in the paranasal sinuses: a review of nasal host defenses.Am J Rhinol. 2008; 22: 13-19Crossref PubMed Scopus (92) Google Scholar It has become clear that proper functioning of the epithelium is essential for suppressing the growth of pathogenic organisms and promoting healthy upper airway physiology. Dysfunction in innate immune expression of host defense molecules has been linked to many airway diseases.3Baines K.J. Simpson J.L. Gibson P.G. Innate immune responses are increased in chronic obstructive pulmonary disease.PLoS One. 2011; 6: e18426Crossref PubMed Scopus (63) Google Scholar, 4Chu H.W. Thaikoottathil J. Rino J.G. Zhang G. Wu Q. Moss T. et al.Function and regulation of SPLUNC1 protein in Mycoplasma infection and allergic inflammation.J Immunol. 2007; 179: 3995-4002Crossref PubMed Scopus (110) Google Scholar, 5Seshadri S. Lin D.C. Rosati M. Carter R.G. Norton J.E. Suh L. et al.Reduced expression of antimicrobial PLUNC proteins in nasal polyp tissues of patients with chronic rhinosinusitis.Allergy. 2012; 67: 920-928Crossref PubMed Scopus (14) Google Scholar, 6Avila P.C. Schleimer R.P. Airway epithelium.in: Allergy and allergic diseases. 2nd ed. Wiley-Blackwell, Hoboken, NJ2008Google Scholar, 7Tieu D.D. Peters A.T. Carter R.G. Suh L. Conley D.B. Chandra R. et al.Evidence for diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis.J Allergy Clin Immunol. 2010; 125: 667-675Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 8Ramanathan Jr., M. Lee W.K. Spannhake E.W. Lane A.P. Th2 cytokines associated with chronic rhinosinusitis with polyps down-regulate the antimicrobial immune function of human sinonasal epithelial cells.Am J Rhinol. 2008; 22: 115-121Crossref PubMed Scopus (99) Google Scholar Surprisingly, the regional distribution of important epithelial-derived antimicrobial proteins secreted into upper airways has not been studied in detail. In this study, we analyzed the expression of select antimicrobial proteins that are known to be secreted into the lining fluids of the upper airways in 2 different anatomical sites of the sinonasal mucosa in healthy human subjects, namely, the inferior region (inferior turbinate [IT]) and the superior region (uncinate tissue [UT]). We chose IT, as it is the proximal point of contact of inhaled air containing particulate matter and potential pathogens. Moreover, studies of chronic rhinosinusitis (CRS) and other diseases of the upper airways routinely use IT tissue as a control tissue to make comparisons with nasal polyps. UT was chosen for its important role in the drainage pathways of maxillary, frontal, and anterior ethmoid sinuses. We chose to study the expression of S100A7 (S100 family), hBD2 (β-defensin family), SPLUNC1 (PLUNC family), and lactoferrin as they represent broad families of antimicrobial proteins, which are either constitutively present or induced by inflammation triggered by pathogens and collectively have antimicrobial effects against a variety of pathogens (bacteria, fungi, and viruses).Sinonasal tissue samples from IT and UT were collected from subjects undergoing surgery to correct noninflammatory conditions, including facial anatomical defects (deformity, trauma, etc), to improve airflow, and during the course of skull base surgery to remove tumors. The subjects were not diagnosed with any upper or lower airway diseases at the time of sample collection. Detailed patient characteristics are provided in Table E1 in this article's Online Repository at www.jacionline.org. The Investigational Review Board of Northwestern University approved all methods for the present study, and all patients provided informed consent. At the time of surgery, tissues and nasal epithelial scraping cells were collected and stored for further analysis. Tissue samples and epithelial cells were analyzed for mRNA expression by using real-time PCR. Protein expression and localization were analyzed by using ELISA and immunohistochemistry, respectively. Alcian blue/periodic acid-Schiff staining was performed to characterize the glandular differences between IT and UT. Details of the methods used can be found in the Online Repository and our other publications.5Seshadri S. Lin D.C. Rosati M. Carter R.G. Norton J.E. Suh L. et al.Reduced expression of antimicrobial PLUNC proteins in nasal polyp tissues of patients with chronic rhinosinusitis.Allergy. 2012; 67: 920-928Crossref PubMed Scopus (14) Google Scholar, 7Tieu D.D. Peters A.T. Carter R.G. Suh L. Conley D.B. Chandra R. et al.Evidence for diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis.J Allergy Clin Immunol. 2010; 125: 667-675Abstract Full Text Full Text PDF PubMed Scopus (38) Google ScholarEarlier studies of S100A7 expression in CRS by our group indicated that S100A7 was differentially expressed in IT and UT tissues.7Tieu D.D. Peters A.T. Carter R.G. Suh L. Conley D.B. Chandra R. et al.Evidence for diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis.J Allergy Clin Immunol. 2010; 125: 667-675Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar To determine regional variations in mRNA expression of this and other innate immune molecules in sinonasal tissues, we performed real-time PCR in a total of 21 samples (10 UT and 11 IT). We found that the mRNA expression of S100A7 and hBD2 was considerably higher in IT tissue than in UT (400- and 80-fold, respectively, Fig 1, A and B). In stark contrast, UT had a substantially higher mRNA expression of SPLUNC1 than did IT tissue (8-fold, Fig 1, E—note log scale). Lactoferrin mRNA expression did not differ between UT and IT (Fig 1, F). Next, we attempted to confirm the mRNA findings at the protein level by using ELISA. In concordance with our mRNA data, the expression of S100A7 and hBD2 proteins was significantly higher in IT than in UT, by 40- and 21-fold, respectively (P < .05) (Fig 1, C and D). Likewise, SPLUNC1 protein was elevated in UT when compared with IT (8-fold, P < .05) (Fig 1, G). In contrast to the mRNA data, lactoferrin protein was 3-fold higher in UT than in IT (Fig 1, H). Because it is well known that sinuses contribute a majority of the respiratory NO production, and because NO plays an antimicrobial role, we also evaluated the expression of various nitric oxide synthases (NOS1, NOS2, and NOS3) in IT and UT (see Fig E1 in this article's Online Repository at www.jacionline.org). We confirmed the previous studies by Lundberg et al,9Lundberg J.O. Farkas-Szallasi T. Weitzberg E. Rinder J. Lidholm J. Anggaard A. et al.High nitric oxide production in human paranasal sinuses.Nat Med. 1995; 1: 370-373Crossref PubMed Scopus (540) Google Scholar showing that the expression of mRNA for NOS2, the major form of NOS in the sinuses involved in the production of NO, was much higher in UT than in IT (22-fold, 292.3 ± 82.8 vs 13.3 ± 9.4 copies/ng total RNA, P = .0006, n = 7-8). Interestingly, we observed that NOS1 (4.7-fold) and NOS3 (1.6-fold) were elevated in IT compared with UT. Taken together, these observations indicate that there is a differential expression of molecules involved in innate host defense in sinonasal tissues taken from different anatomical sites of healthy human subjects.We confirmed our previous observation that S100A7 was primarily expressed in the mucosal epithelium with modest staining in submucosal glands, whereas SPLUNC1 was highly expressed in the submucosal glands with minimal presence in the mucosal epithelium (see Fig E2 in this article's Online Repository at www.jacionline.org).5Seshadri S. Lin D.C. Rosati M. Carter R.G. Norton J.E. Suh L. et al.Reduced expression of antimicrobial PLUNC proteins in nasal polyp tissues of patients with chronic rhinosinusitis.Allergy. 2012; 67: 920-928Crossref PubMed Scopus (14) Google Scholar, 7Tieu D.D. Peters A.T. Carter R.G. Suh L. Conley D.B. Chandra R. et al.Evidence for diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis.J Allergy Clin Immunol. 2010; 125: 667-675Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar To further confirm the immunohistochemistry data indicating high expression levels of S100A7 in the surface epithelium, we performed ELISA with isolated nasal epithelial cells from IT and UT tissues. In agreement with our ELISA data in whole tissue homogenates, we found that IT epithelial cells had a 12-fold higher S100A7 protein expression than did UT epithelial cells (Fig 1, I). In contrast, the expression of SPLUNC1 in epithelial cells from IT and UT was not different (Fig 1, J). This suggests that the regional variation of SPLUNC1 expression in tissue samples may be caused by variability in expression in glands rather than in mucosal epithelium (see below). These findings also suggest that the underlying molecular mechanism responsible for the regional variation of S100A7 may differ from the differential transcriptional expression mechanism of SPLUNC1.To further understand the regional variations between IT and UT tissues in the expression of host defense molecules that are largely derived from submucosal glands, specifically SPLUNC1 and lactoferrin, we assessed the relative expression of these molecules in both serous and mucous cells within glands from both locations. In initial studies, we stained both tissues with mucous (alcian blue) and serous (periodic acid-Schiff) stains. We failed to observe any appreciable differences in the number or proportion of serous and mucous cells in IT when compared to UT tissues (Fig 1, K and L; see Fig E3 in this article's Online Repository at www.jacionline.org). These experiments indicate that the regional variation of host defense molecules is not explained by regional variation in either submucosal gland density or proportions of serous and mucous cells within glands. We must caution that while there is no appreciable difference in the number of glands based on microscopic observation, it remains possible that there are differences in the volume of glands between the IT and UT tissues.A pictorial summary of our findings is shown in Fig 2. Our finding of differential expression of innate immune proteins between IT and UT is unexpected because these tissues have been used interchangeably to study gene expression levels and as control tissue to make comparisons with diseased tissues such as nasal polyps in the case of CRS. A recent report from Laudien et al10Laudien M. Dressel S. Harder J. Glaser R. Differential expression pattern of antimicrobial peptides in nasal mucosa and secretion.Rhinology. 2011; 49: 107-111PubMed Google Scholar compared the expression of host defense molecules between the nasal vestibule (anterior part of the nasal cavity) and the IT region of the proper nasal mucosa. The findings of differential expression of some of the molecules tested in their study between the nasal vestibule and IT are interesting, though not surprising, because it is known that epithelial cells of the nasal vestibule are similar to keratinocytes (stratified squamous), which are very different from respiratory epithelial cells (pseudostratified columnar).Fig 2Regional differences in the expression of innate host defense molecules in sinonasal mucosa.View Large Image Figure ViewerDownload Hi-res image Download (PPT)This study demonstrates that the expression of some innate host defense molecules in various regional tissues is quite variable, with expression of some glandular proteins (lactoferrin and SPLUNC1) greater in UT than in IT. Interestingly, we initially used the mucin MUC5B as a marker of mucous cells, but found a differential expression similar to that of SPLUNC1—that is, high levels in UT and much lower levels in IT (6181 ± 1993 vs 2147 ± 411 copies/ng of total RNA, P = .03). This supports the concept that submucosal glands near the UT are a much richer source of a number of host defense molecules than are corresponding glands in the IT region. The apparent differences that we observe could be due to the different embryonic origins of these tissues (maxilloturbinal [IT] vs ethmoturbinal [UT]). As a result of different embryonic origins, these tissues could be governed by ontologically different gene regulation pathways. Alternatively, regionally dependent environmental exposures may also be a cause or factor in the differential expression observed. Finally, as UT is located at a point of drainage of various sinuses, it is tempting to speculate that the increased expression of host defense molecules such as SPLUNC1, lactoferrin, and MUC5B in UT may be important for additional host defense in sinus drainage pathways. In support of this hypothesis, there is emerging evidence indicating that SPLUNC1 has surfactant properties and may be involved in biophysical aspects of mucociliary clearance.11McGillivary G. Bakaletz L.O. The multifunctional host defense peptide SPLUNC1 is critical for homeostasis of the mammalian upper airway.PLoS One. 2010; 5: e13224Crossref PubMed Scopus (50) Google Scholar Our data also suggest that there may potentially be different microbiota and/or susceptibilities for microbial colonization in the proximal (IT) and distal (UT) areas of the sinonasal mucosa, with UT having a more profound expression of submucosal gland–derived antimicrobial proteins.In conclusion, our findings indicate anatomic site-specific expression of antimicrobial proteins in sinonasal tissues, suggesting the possibility of specialized regional functions of these proteins within the sinonasal cavity. Also, our findings of different intrinsic baseline levels of gene expression in IT and UT suggest that a careful selection of the site of control tissue is needed when studying host defense molecules. Nasal mucosal epithelial cells form the first line of defense by maintaining a barrier to restrict potentially harmful airborne substances and pathogens. To aid in host defense, specialized epithelial cells in submucosal glands as well as on the mucosal surface secrete mucous that helps to immobilize pathogens and other harmful substances. Beneath this mucous blanket resides an aqueous surface liquid layer that allows proper ciliary function to clear the mucous and entrapped pathogens. In concert with the maintenance of a physical barrier, airway epithelium secretes dozens of antimicrobial peptides and proteins that become incorporated into the mucous and aqueous lining fluids.1Kato A. Schleimer R.P. Beyond inflammation: airway epithelial cells are at the interface of innate and adaptive immunity.Curr Opin Immunol. 2007; 19: 711-720Crossref PubMed Scopus (261) Google Scholar, 2Ooi E.H. Wormald P.J. Tan L.W. Innate immunity in the paranasal sinuses: a review of nasal host defenses.Am J Rhinol. 2008; 22: 13-19Crossref PubMed Scopus (92) Google Scholar It has become clear that proper functioning of the epithelium is essential for suppressing the growth of pathogenic organisms and promoting healthy upper airway physiology. Dysfunction in innate immune expression of host defense molecules has been linked to many airway diseases.3Baines K.J. Simpson J.L. Gibson P.G. Innate immune responses are increased in chronic obstructive pulmonary disease.PLoS One. 2011; 6: e18426Crossref PubMed Scopus (63) Google Scholar, 4Chu H.W. Thaikoottathil J. Rino J.G. Zhang G. Wu Q. Moss T. et al.Function and regulation of SPLUNC1 protein in Mycoplasma infection and allergic inflammation.J Immunol. 2007; 179: 3995-4002Crossref PubMed Scopus (110) Google Scholar, 5Seshadri S. Lin D.C. Rosati M. Carter R.G. Norton J.E. Suh L. et al.Reduced expression of antimicrobial PLUNC proteins in nasal polyp tissues of patients with chronic rhinosinusitis.Allergy. 2012; 67: 920-928Crossref PubMed Scopus (14) Google Scholar, 6Avila P.C. Schleimer R.P. Airway epithelium.in: Allergy and allergic diseases. 2nd ed. Wiley-Blackwell, Hoboken, NJ2008Google Scholar, 7Tieu D.D. Peters A.T. Carter R.G. Suh L. Conley D.B. Chandra R. et al.Evidence for diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis.J Allergy Clin Immunol. 2010; 125: 667-675Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar, 8Ramanathan Jr., M. Lee W.K. Spannhake E.W. Lane A.P. Th2 cytokines associated with chronic rhinosinusitis with polyps down-regulate the antimicrobial immune function of human sinonasal epithelial cells.Am J Rhinol. 2008; 22: 115-121Crossref PubMed Scopus (99) Google Scholar Surprisingly, the regional distribution of important epithelial-derived antimicrobial proteins secreted into upper airways has not been studied in detail. In this study, we analyzed the expression of select antimicrobial proteins that are known to be secreted into the lining fluids of the upper airways in 2 different anatomical sites of the sinonasal mucosa in healthy human subjects, namely, the inferior region (inferior turbinate [IT]) and the superior region (uncinate tissue [UT]). We chose IT, as it is the proximal point of contact of inhaled air containing particulate matter and potential pathogens. Moreover, studies of chronic rhinosinusitis (CRS) and other diseases of the upper airways routinely use IT tissue as a control tissue to make comparisons with nasal polyps. UT was chosen for its important role in the drainage pathways of maxillary, frontal, and anterior ethmoid sinuses. We chose to study the expression of S100A7 (S100 family), hBD2 (β-defensin family), SPLUNC1 (PLUNC family), and lactoferrin as they represent broad families of antimicrobial proteins, which are either constitutively present or induced by inflammation triggered by pathogens and collectively have antimicrobial effects against a variety of pathogens (bacteria, fungi, and viruses). Sinonasal tissue samples from IT and UT were collected from subjects undergoing surgery to correct noninflammatory conditions, including facial anatomical defects (deformity, trauma, etc), to improve airflow, and during the course of skull base surgery to remove tumors. The subjects were not diagnosed with any upper or lower airway diseases at the time of sample collection. Detailed patient characteristics are provided in Table E1 in this article's Online Repository at www.jacionline.org. The Investigational Review Board of Northwestern University approved all methods for the present study, and all patients provided informed consent. At the time of surgery, tissues and nasal epithelial scraping cells were collected and stored for further analysis. Tissue samples and epithelial cells were analyzed for mRNA expression by using real-time PCR. Protein expression and localization were analyzed by using ELISA and immunohistochemistry, respectively. Alcian blue/periodic acid-Schiff staining was performed to characterize the glandular differences between IT and UT. Details of the methods used can be found in the Online Repository and our other publications.5Seshadri S. Lin D.C. Rosati M. Carter R.G. Norton J.E. Suh L. et al.Reduced expression of antimicrobial PLUNC proteins in nasal polyp tissues of patients with chronic rhinosinusitis.Allergy. 2012; 67: 920-928Crossref PubMed Scopus (14) Google Scholar, 7Tieu D.D. Peters A.T. Carter R.G. Suh L. Conley D.B. Chandra R. et al.Evidence for diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis.J Allergy Clin Immunol. 2010; 125: 667-675Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar Earlier studies of S100A7 expression in CRS by our group indicated that S100A7 was differentially expressed in IT and UT tissues.7Tieu D.D. Peters A.T. Carter R.G. Suh L. Conley D.B. Chandra R. et al.Evidence for diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis.J Allergy Clin Immunol. 2010; 125: 667-675Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar To determine regional variations in mRNA expression of this and other innate immune molecules in sinonasal tissues, we performed real-time PCR in a total of 21 samples (10 UT and 11 IT). We found that the mRNA expression of S100A7 and hBD2 was considerably higher in IT tissue than in UT (400- and 80-fold, respectively, Fig 1, A and B). In stark contrast, UT had a substantially higher mRNA expression of SPLUNC1 than did IT tissue (8-fold, Fig 1, E—note log scale). Lactoferrin mRNA expression did not differ between UT and IT (Fig 1, F). Next, we attempted to confirm the mRNA findings at the protein level by using ELISA. In concordance with our mRNA data, the expression of S100A7 and hBD2 proteins was significantly higher in IT than in UT, by 40- and 21-fold, respectively (P < .05) (Fig 1, C and D). Likewise, SPLUNC1 protein was elevated in UT when compared with IT (8-fold, P < .05) (Fig 1, G). In contrast to the mRNA data, lactoferrin protein was 3-fold higher in UT than in IT (Fig 1, H). Because it is well known that sinuses contribute a majority of the respiratory NO production, and because NO plays an antimicrobial role, we also evaluated the expression of various nitric oxide synthases (NOS1, NOS2, and NOS3) in IT and UT (see Fig E1 in this article's Online Repository at www.jacionline.org). We confirmed the previous studies by Lundberg et al,9Lundberg J.O. Farkas-Szallasi T. Weitzberg E. Rinder J. Lidholm J. Anggaard A. et al.High nitric oxide production in human paranasal sinuses.Nat Med. 1995; 1: 370-373Crossref PubMed Scopus (540) Google Scholar showing that the expression of mRNA for NOS2, the major form of NOS in the sinuses involved in the production of NO, was much higher in UT than in IT (22-fold, 292.3 ± 82.8 vs 13.3 ± 9.4 copies/ng total RNA, P = .0006, n = 7-8). Interestingly, we observed that NOS1 (4.7-fold) and NOS3 (1.6-fold) were elevated in IT compared with UT. Taken together, these observations indicate that there is a differential expression of molecules involved in innate host defense in sinonasal tissues taken from different anatomical sites of healthy human subjects. We confirmed our previous observation that S100A7 was primarily expressed in the mucosal epithelium with modest staining in submucosal glands, whereas SPLUNC1 was highly expressed in the submucosal glands with minimal presence in the mucosal epithelium (see Fig E2 in this article's Online Repository at www.jacionline.org).5Seshadri S. Lin D.C. Rosati M. Carter R.G. Norton J.E. Suh L. et al.Reduced expression of antimicrobial PLUNC proteins in nasal polyp tissues of patients with chronic rhinosinusitis.Allergy. 2012; 67: 920-928Crossref PubMed Scopus (14) Google Scholar, 7Tieu D.D. Peters A.T. Carter R.G. Suh L. Conley D.B. Chandra R. et al.Evidence for diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis.J Allergy Clin Immunol. 2010; 125: 667-675Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar To further confirm the immunohistochemistry data indicating high expression levels of S100A7 in the surface epithelium, we performed ELISA with isolated nasal epithelial cells from IT and UT tissues. In agreement with our ELISA data in whole tissue homogenates, we found that IT epithelial cells had a 12-fold higher S100A7 protein expression than did UT epithelial cells (Fig 1, I). In contrast, the expression of SPLUNC1 in epithelial cells from IT and UT was not different (Fig 1, J). This suggests that the regional variation of SPLUNC1 expression in tissue samples may be caused by variability in expression in glands rather than in mucosal epithelium (see below). These findings also suggest that the underlying molecular mechanism responsible for the regional variation of S100A7 may differ from the differential transcriptional expression mechanism of SPLUNC1. To further understand the regional variations between IT and UT tissues in the expression of host defense molecules that are largely derived from submucosal glands, specifically SPLUNC1 and lactoferrin, we assessed the relative expression of these molecules in both serous and mucous cells within glands from both locations. In initial studies, we stained both tissues with mucous (alcian blue) and serous (periodic acid-Schiff) stains. We failed to observe any appreciable differences in the number or proportion of serous and mucous cells in IT when compared to UT tissues (Fig 1, K and L; see Fig E3 in this article's Online Repository at www.jacionline.org). These experiments indicate that the regional variation of host defense molecules is not explained by regional variation in either submucosal gland density or proportions of serous and mucous cells within glands. We must caution that while there is no appreciable difference in the number of glands based on microscopic observation, it remains possible that there are differences in the volume of glands between the IT and UT tissues. A pictorial summary of our findings is shown in Fig 2. Our finding of differential expression of innate immune proteins between IT and UT is unexpected because these tissues have been used interchangeably to study gene expression levels and as control tissue to make comparisons with diseased tissues such as nasal polyps in the case of CRS. A recent report from Laudien et al10Laudien M. Dressel S. Harder J. Glaser R. Differential expression pattern of antimicrobial peptides in nasal mucosa and secretion.Rhinology. 2011; 49: 107-111PubMed Google Scholar compared the expression of host defense molecules between the nasal vestibule (anterior part of the nasal cavity) and the IT region of the proper nasal mucosa. The findings of differential expression of some of the molecules tested in their study between the nasal vestibule and IT are interesting, though not surprising, because it is known that epithelial cells of the nasal vestibule are similar to keratinocytes (stratified squamous), which are very different from respiratory epithelial cells (pseudostratified columnar). This study demonstrates that the expression of some innate host defense molecules in various regional tissues is quite variable, with expression of some glandular proteins (lactoferrin and SPLUNC1) greater in UT than in IT. Interestingly, we initially used the mucin MUC5B as a marker of mucous cells, but found a differential expression similar to that of SPLUNC1—that is, high levels in UT and much lower levels in IT (6181 ± 1993 vs 2147 ± 411 copies/ng of total RNA, P = .03). This supports the concept that submucosal glands near the UT are a much richer source of a number of host defense molecules than are corresponding glands in the IT region. The apparent differences that we observe could be due to the different embryonic origins of these tissues (maxilloturbinal [IT] vs ethmoturbinal [UT]). As a result of different embryonic origins, these tissues could be governed by ontologically different gene regulation pathways. Alternatively, regionally dependent environmental exposures may also be a cause or factor in the differential expression observed. Finally, as UT is located at a point of drainage of various sinuses, it is tempting to speculate that the increased expression of host defense molecules such as SPLUNC1, lactoferrin, and MUC5B in UT may be important for additional host defense in sinus drainage pathways. In support of this hypothesis, there is emerging evidence indicating that SPLUNC1 has surfactant properties and may be involved in biophysical aspects of mucociliary clearance.11McGillivary G. Bakaletz L.O. The multifunctional host defense peptide SPLUNC1 is critical for homeostasis of the mammalian upper airway.PLoS One. 2010; 5: e13224Crossref PubMed Scopus (50) Google Scholar Our data also suggest that there may potentially be different microbiota and/or susceptibilities for microbial colonization in the proximal (IT) and distal (UT) areas of the sinonasal mucosa, with UT having a more profound expression of submucosal gland–derived antimicrobial proteins. In conclusion, our findings indicate anatomic site-specific expression of antimicrobial proteins in sinonasal tissues, suggesting the possibility of specialized regional functions of these proteins within the sinonasal cavity. Also, our findings of different intrinsic baseline levels of gene expression in IT and UT suggest that a careful selection of the site of control tissue is needed when studying host defense molecules. We thank Ms Jacqi Schaffer for assistance in producing Fig 2. MethodsReal-time PCRTotal RNA from sinonasal tissue was extracted by using QIAzol (Qiagen, Valencia, Calif) using RNeasy purification system according to manufacturer's instructions. DNA contamination was eliminated by treatment of RNA with DNase I during purification. Purified RNA was assessed with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif) by using a RNA 6000 Nano LabChip (Agilent Technologies). One microgram of RNA was converted to single-strand cDNA by using SuperScript II reverse transcriptase system (Invitrogen, Carlsbad, Calif) using random primers. Semi-quantitative real-time RT-PCR was performed with a TaqMan method by using an Applied Biosystems 7500 Sequence Detection System (Applied Biosystems, Foster City, Calif) in 15 μL reactions (7.5 μL of 2× TaqMan Gene Expression Master mix [Applied Biosystems], 0.75 μL of 20× primer/probe mix, and 5 μL of 2 ng/μL cDNA). All primers/probes were purchased from Applied Biosystems. Primers for S100A7 have been described previously.E1Richer S.L. Truong-Tran A.Q. Conley D.B. Carter R. Vermylen D. Grammer L.C. et al.Epithelial genes in chronic rhinosinusitis with and without nasal polyps.Am J Rhinol. 2008; 22: 228-234Crossref PubMed Scopus (66) Google Scholar Median expression of GUSB (Human β-glucuronidase endogenous control, PN; 4326320E) was used for normalizing gene expression. Exact copy number of the target genes was determined by running serial dilutions of quantified PCR product of the target gene as standards in the PCR reaction. Target gene expression was expressed as copies/ng of total RNA.Extraction of proteins from sinus tissue and nasal scraping cellsTissue proteins were extracted as described in Seshadri et al.E2Seshadri S. Lin D.C. Rosati M. Carter R.G. Norton J.E. Suh L. et al.Reduced expression of antimicrobial PLUNC proteins in nasal polyp tissues of patients with chronic rhinosinusitis.Allergy. 2012; 67: 920-928Crossref PubMed Scopus (82) Google Scholar Briefly, tissues were cut into small pieces and resuspended in PBS containing 0.05% Tween 20 supplemented with a protease inhibitor cocktail (Sigma-Aldrich, St Louis, Mo). Tissues were lysed in bullet blender (Next Advance, Averill Park, NY) with 1.6 mm stainless steel beads for 4 minutes twice at setting 7. Tissue lysates were centrifuged at 1968g to clear the debris. Tissue lysates were stored at −20°C until further use. Nasal scraping cells were collected by scraping tissues with Rhinoprobe. Nasal scraping cells were suspended in RPMI1640 medium. Cells were centrifuged at 5000g for 5 minutes. Pelleted cells were washed with PBS and centrifuged again at 5000g for an additional 5 minutes. Pelleted cells were lysed by vortexing in PBS supplemented with 0.05% Tween 20, protease, and phosphatase inhibitor (Sigma). Cell lysates were spun at 5000g for 5 minutes to remove debris. Clarified cell lysates were used for S100A7 and SPLUNC1 ELISA.ImmunohistochemistryImmunohistochemistry was performed as described in Seshadri et alE2Seshadri S. Lin D.C. Rosati M. Carter R.G. Norton J.E. Suh L. et al.Reduced expression of antimicrobial PLUNC proteins in nasal polyp tissues of patients with chronic rhinosinusitis.Allergy. 2012; 67: 920-928Crossref PubMed Scopus (82) Google Scholar (SPLUNC1) and Tieu et alE3Tieu D.D. Peters A.T. Carter R.G. Suh L. Conley D.B. Chandra R. et al.Evidence for diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis.J Allergy Clin Immunol. 2010; 125: 667-675Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar (S100A7). Sections stained for alcian blue/periodic acid-Schiff reagents were deparaffinized, rehydrated, and stained sequentially with alcian blue (Sigma-Aldrich; St Louis, Mo), periodic acid (Electron Microscopy Sciences; Hatfield, Pa), and Schiff reagent (Richard-Allan Scientific, Kalamazoo, Mich). Glandular quantitation of alcian blue/periodic acid-Schiff sections was performed by 2 independent observers in 10 randomly chosen fields by using an Olympus IX71 Inverted Microscope at a magnification of 200× and a MicroFire AR digital microscope camera (Optronics, Goleta, Calif). Each section was blinded, and subject diagnosis was unknown to the observers.ELISAFig E2Immunohistochemical staining for SPLUNC1 and S100A7 in sinonasal tissue. SPLUNC1 (A) and S100A7 (B) immunostaining in uncinate of a control subject. Representative of at least 3 donors per group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Fig E3Alcian blue/periodic acid-Schiff (AB/PAS) staining in sinonasal tissues. AB/PAS staining in uncinate (A) and inferior turbinate (B) of a control subject. Representative of 5 donors per group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Subject characteristicsPCRUncinateITNo. of subjects10 (3 M)11 (9 M)Age (y), median (range)42 (16-62)37 (27-77)Tissues-ELISAUncinateITS100A7 No. of subjects9 (4 M)9 (7 M) Age (y), median (range)46 (16-59)40 (25-59)Beta defensin-2 No. of subjects11 (7 M)16 (13 M) Age (y), median (range)45 (16-59)40 (19-62)SPLUNC1 No. of subjects10 (4 M)12 (8 M) Age (y), median (range)52.5 (16-72)36 (17-72)Lactoferrin No. of subjects7 (5 M)10 (7 M) Age (y), median (range)45 (19-62)40 (17-62)NECs-ELISAUncinateITS100A7 No. of subjects14 (10 M)16 (13 M) Age (y), median (range)52 (19-72)48 (19-72)SPLUNC1 No. of subjects8 (7 M)7 (5 M) Age (y), median (range)50.5 (19-72)52 (19-72)Alcian blue/PASUncinateIT No. of subjects5 (4 M)5 (3 M) Age (y), median (range)49 (19-62)35 (16-62)IHC, Immunohistochemistry; M, male; NECs, nasal epithelial cells; PAS, periodic acid-Schiff. Open table in a new tab Real-time PCRTotal RNA from sinonasal tissue was extracted by using QIAzol (Qiagen, Valencia, Calif) using RNeasy purification system according to manufacturer's instructions. DNA contamination was eliminated by treatment of RNA with DNase I during purification. Purified RNA was assessed with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif) by using a RNA 6000 Nano LabChip (Agilent Technologies). One microgram of RNA was converted to single-strand cDNA by using SuperScript II reverse transcriptase system (Invitrogen, Carlsbad, Calif) using random primers. Semi-quantitative real-time RT-PCR was performed with a TaqMan method by using an Applied Biosystems 7500 Sequence Detection System (Applied Biosystems, Foster City, Calif) in 15 μL reactions (7.5 μL of 2× TaqMan Gene Expression Master mix [Applied Biosystems], 0.75 μL of 20× primer/probe mix, and 5 μL of 2 ng/μL cDNA). All primers/probes were purchased from Applied Biosystems. Primers for S100A7 have been described previously.E1Richer S.L. Truong-Tran A.Q. Conley D.B. Carter R. Vermylen D. Grammer L.C. et al.Epithelial genes in chronic rhinosinusitis with and without nasal polyps.Am J Rhinol. 2008; 22: 228-234Crossref PubMed Scopus (66) Google Scholar Median expression of GUSB (Human β-glucuronidase endogenous control, PN; 4326320E) was used for normalizing gene expression. Exact copy number of the target genes was determined by running serial dilutions of quantified PCR product of the target gene as standards in the PCR reaction. Target gene expression was expressed as copies/ng of total RNA. Total RNA from sinonasal tissue was extracted by using QIAzol (Qiagen, Valencia, Calif) using RNeasy purification system according to manufacturer's instructions. DNA contamination was eliminated by treatment of RNA with DNase I during purification. Purified RNA was assessed with Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, Calif) by using a RNA 6000 Nano LabChip (Agilent Technologies). One microgram of RNA was converted to single-strand cDNA by using SuperScript II reverse transcriptase system (Invitrogen, Carlsbad, Calif) using random primers. Semi-quantitative real-time RT-PCR was performed with a TaqMan method by using an Applied Biosystems 7500 Sequence Detection System (Applied Biosystems, Foster City, Calif) in 15 μL reactions (7.5 μL of 2× TaqMan Gene Expression Master mix [Applied Biosystems], 0.75 μL of 20× primer/probe mix, and 5 μL of 2 ng/μL cDNA). All primers/probes were purchased from Applied Biosystems. Primers for S100A7 have been described previously.E1Richer S.L. Truong-Tran A.Q. Conley D.B. Carter R. Vermylen D. Grammer L.C. et al.Epithelial genes in chronic rhinosinusitis with and without nasal polyps.Am J Rhinol. 2008; 22: 228-234Crossref PubMed Scopus (66) Google Scholar Median expression of GUSB (Human β-glucuronidase endogenous control, PN; 4326320E) was used for normalizing gene expression. Exact copy number of the target genes was determined by running serial dilutions of quantified PCR product of the target gene as standards in the PCR reaction. Target gene expression was expressed as copies/ng of total RNA. Extraction of proteins from sinus tissue and nasal scraping cellsTissue proteins were extracted as described in Seshadri et al.E2Seshadri S. Lin D.C. Rosati M. Carter R.G. Norton J.E. Suh L. et al.Reduced expression of antimicrobial PLUNC proteins in nasal polyp tissues of patients with chronic rhinosinusitis.Allergy. 2012; 67: 920-928Crossref PubMed Scopus (82) Google Scholar Briefly, tissues were cut into small pieces and resuspended in PBS containing 0.05% Tween 20 supplemented with a protease inhibitor cocktail (Sigma-Aldrich, St Louis, Mo). Tissues were lysed in bullet blender (Next Advance, Averill Park, NY) with 1.6 mm stainless steel beads for 4 minutes twice at setting 7. Tissue lysates were centrifuged at 1968g to clear the debris. Tissue lysates were stored at −20°C until further use. Nasal scraping cells were collected by scraping tissues with Rhinoprobe. Nasal scraping cells were suspended in RPMI1640 medium. Cells were centrifuged at 5000g for 5 minutes. Pelleted cells were washed with PBS and centrifuged again at 5000g for an additional 5 minutes. Pelleted cells were lysed by vortexing in PBS supplemented with 0.05% Tween 20, protease, and phosphatase inhibitor (Sigma). Cell lysates were spun at 5000g for 5 minutes to remove debris. Clarified cell lysates were used for S100A7 and SPLUNC1 ELISA. Tissue proteins were extracted as described in Seshadri et al.E2Seshadri S. Lin D.C. Rosati M. Carter R.G. Norton J.E. Suh L. et al.Reduced expression of antimicrobial PLUNC proteins in nasal polyp tissues of patients with chronic rhinosinusitis.Allergy. 2012; 67: 920-928Crossref PubMed Scopus (82) Google Scholar Briefly, tissues were cut into small pieces and resuspended in PBS containing 0.05% Tween 20 supplemented with a protease inhibitor cocktail (Sigma-Aldrich, St Louis, Mo). Tissues were lysed in bullet blender (Next Advance, Averill Park, NY) with 1.6 mm stainless steel beads for 4 minutes twice at setting 7. Tissue lysates were centrifuged at 1968g to clear the debris. Tissue lysates were stored at −20°C until further use. Nasal scraping cells were collected by scraping tissues with Rhinoprobe. Nasal scraping cells were suspended in RPMI1640 medium. Cells were centrifuged at 5000g for 5 minutes. Pelleted cells were washed with PBS and centrifuged again at 5000g for an additional 5 minutes. Pelleted cells were lysed by vortexing in PBS supplemented with 0.05% Tween 20, protease, and phosphatase inhibitor (Sigma). Cell lysates were spun at 5000g for 5 minutes to remove debris. Clarified cell lysates were used for S100A7 and SPLUNC1 ELISA. ImmunohistochemistryImmunohistochemistry was performed as described in Seshadri et alE2Seshadri S. Lin D.C. Rosati M. Carter R.G. Norton J.E. Suh L. et al.Reduced expression of antimicrobial PLUNC proteins in nasal polyp tissues of patients with chronic rhinosinusitis.Allergy. 2012; 67: 920-928Crossref PubMed Scopus (82) Google Scholar (SPLUNC1) and Tieu et alE3Tieu D.D. Peters A.T. Carter R.G. Suh L. Conley D.B. Chandra R. et al.Evidence for diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis.J Allergy Clin Immunol. 2010; 125: 667-675Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar (S100A7). Sections stained for alcian blue/periodic acid-Schiff reagents were deparaffinized, rehydrated, and stained sequentially with alcian blue (Sigma-Aldrich; St Louis, Mo), periodic acid (Electron Microscopy Sciences; Hatfield, Pa), and Schiff reagent (Richard-Allan Scientific, Kalamazoo, Mich). Glandular quantitation of alcian blue/periodic acid-Schiff sections was performed by 2 independent observers in 10 randomly chosen fields by using an Olympus IX71 Inverted Microscope at a magnification of 200× and a MicroFire AR digital microscope camera (Optronics, Goleta, Calif). Each section was blinded, and subject diagnosis was unknown to the observers. Immunohistochemistry was performed as described in Seshadri et alE2Seshadri S. Lin D.C. Rosati M. Carter R.G. Norton J.E. Suh L. et al.Reduced expression of antimicrobial PLUNC proteins in nasal polyp tissues of patients with chronic rhinosinusitis.Allergy. 2012; 67: 920-928Crossref PubMed Scopus (82) Google Scholar (SPLUNC1) and Tieu et alE3Tieu D.D. Peters A.T. Carter R.G. Suh L. Conley D.B. Chandra R. et al.Evidence for diminished levels of epithelial psoriasin and calprotectin in chronic rhinosinusitis.J Allergy Clin Immunol. 2010; 125: 667-675Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar (S100A7). Sections stained for alcian blue/periodic acid-Schiff reagents were deparaffinized, rehydrated, and stained sequentially with alcian blue (Sigma-Aldrich; St Louis, Mo), periodic acid (Electron Microscopy Sciences; Hatfield, Pa), and Schiff reagent (Richard-Allan Scientific, Kalamazoo, Mich). Glandular quantitation of alcian blue/periodic acid-Schiff sections was performed by 2 independent observers in 10 randomly chosen fields by using an Olympus IX71 Inverted Microscope at a magnification of 200× and a MicroFire AR digital microscope camera (Optronics, Goleta, Calif). Each section was blinded, and subject diagnosis was unknown to the observers. ELISAFig E3Alcian blue/periodic acid-Schiff (AB/PAS) staining in sinonasal tissues. AB/PAS staining in uncinate (A) and inferior turbinate (B) of a control subject. Representative of 5 donors per group.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table E1Subject characteristicsPCRUncinateITNo. of subjects10 (3 M)11 (9 M)Age (y), median (range)42 (16-62)37 (27-77)Tissues-ELISAUncinateITS100A7 No. of subjects9 (4 M)9 (7 M) Age (y), median (range)46 (16-59)40 (25-59)Beta defensin-2 No. of subjects11 (7 M)16 (13 M) Age (y), median (range)45 (16-59)40 (19-62)SPLUNC1 No. of subjects10 (4 M)12 (8 M) Age (y), median (range)52.5 (16-72)36 (17-72)Lactoferrin No. of subjects7 (5 M)10 (7 M) Age (y), median (range)45 (19-62)40 (17-62)NECs-ELISAUncinateITS100A7 No. of subjects14 (10 M)16 (13 M) Age (y), median (range)52 (19-72)48 (19-72)SPLUNC1 No. of subjects8 (7 M)7 (5 M) Age (y), median (range)50.5 (19-72)52 (19-72)Alcian blue/PASUncinateIT No. of subjects5 (4 M)5 (3 M) Age (y), median (range)49 (19-62)35 (16-62)IHC, Immunohistochemistry; M, male; NECs, nasal epithelial cells; PAS, periodic acid-Schiff. Open table in a new tab IHC, Immunohistochemistry; M, male; NECs, nasal epithelial cells; PAS, periodic acid-Schiff.
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