Basophils are elevated in nasal polyps of patients with chronic rhinosinusitis without aspirin sensitivity

Abstract

Chronic rhinosinusitis (CRS) is a common chronic disease with significant burden and effect on the quality of life of affected individuals, characterized by inflammation of the nasal mucosa and paranasal sinuses. Patients can be subdivided into CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). In addition, aspirin exacerbated respiratory disease (AERD) is a subgroup of CRSwNP characterized by the triad of CRSwNP, asthma, and hypersensitivity to aspirin or nonsteroidal antiinflammatory drugs. Eosinophilia is evident in the nasal mucosa and submucosa of nasal polyps (NPs) from patients in Europe and the United States, but less so in Asian countries such as China or Japan.1Zhang N. Van Zele T. Perez-Novo C. Van Bruaene N. Holtappels G. DeRuyck N. et al.Different types of T-effector cells orchestrate mucosal inflammation in chronic sinus disease.J Allergy Clin Immunol. 2008; 122: 961-968Google Scholar In this study we attempted, for the first time, to evaluate the role of another important inflammatory leukocyte, the basophil, in CRS. Basophils are granulocytes found mainly in the circulation and are known to have a role in allergic diseases, parasite expulsion, and immunity against ectoparasites.2Schroeder J.T. Basophils: emerging roles in the pathogenesis of allergic disease.Immunol Rev. 2011; 242: 144-160Google Scholar Basophil numbers are elevated in the bronchial mucosa and submucosa of asthmatic patients, the nasal submucosa in patients with allergic rhinitis, and in the skin of those with multiple inflammatory dermatologic conditions, including eczema and urticaria.2Schroeder J.T. Basophils: emerging roles in the pathogenesis of allergic disease.Immunol Rev. 2011; 242: 144-160Google Scholar, 3Nouri-Aria K.T. Irani A.M. Jacobson M.R. O'Brien F. Varga E.M. Till S.J. et al.Basophil recruitment and IL-4 production during human allergen-induced late asthma.J Allergy Clin Immunol. 2001; 108: 205-211Google Scholar, 4Gauvreau G.M. Lee J.M. Watson R.M. Irani A.M. Schwartz L.B. O'Byrne P.M. Increased numbers of both airway basophils and mast cells in sputum after allergen inhalation challenge of atopic asthmatics.Am J Respir Crit Care Med. 2000; 161: 1473-1478Google Scholar, 5Wilson D.R. Irani A.M. Walker S.M. Jacobson M.R. Mackay I.S. Schwartz L.B. et al.Grass pollen immunotherapy inhibits seasonal increases in basophils and eosinophils in the nasal epithelium.Clin Exp Allergy. 2001; 31: 1705-1713Google ScholarAlthough CRSwNP is a highly TH2-biased disease, making basophil involvement likely, there are no reports of basophils in CRS. In the current study we used 2D7, a mouse mAb against a basophil-specific intermediate form of major basic protein,6Plager D.A. Weiss E.A. Kephart G.M. Mocharla R.M. Matsumoto R. Checkel J.L. et al.Identification of basophils by a mAb directed against pro-major basic protein 1.J Allergy Clin Immunol. 2006; 117: 626-634Google Scholar to detect basophils in uncinate tissue (UT) or polyp tissue obtained during surgery from patients with CRS and control subjects as described previously.7Takabayashi T. Kato A. Peters A.T. Suh L.A. Carter R. Norton J. et al.Glandular mast cells with distinct phenotype are highly elevated in chronic rhinosinusitis with nasal polyps.J Allergy Clin Immunol. 2012; 130: 410-420.e5Google Scholar This antibody is shown to be a specific marker for basophils and does not recognize any other cell type.6Plager D.A. Weiss E.A. Kephart G.M. Mocharla R.M. Matsumoto R. Checkel J.L. et al.Identification of basophils by a mAb directed against pro-major basic protein 1.J Allergy Clin Immunol. 2006; 117: 626-634Google Scholar We used the immunohistochemistry protocol detailed in previous studies using 2D7.3Nouri-Aria K.T. Irani A.M. Jacobson M.R. O'Brien F. Varga E.M. Till S.J. et al.Basophil recruitment and IL-4 production during human allergen-induced late asthma.J Allergy Clin Immunol. 2001; 108: 205-211Google Scholar, 5Wilson D.R. Irani A.M. Walker S.M. Jacobson M.R. Mackay I.S. Schwartz L.B. et al.Grass pollen immunotherapy inhibits seasonal increases in basophils and eosinophils in the nasal epithelium.Clin Exp Allergy. 2001; 31: 1705-1713Google Scholar Mucosa and submucosa in the tissue biopsies were delineated, and the total number of positively stained cells in each field was counted in 50 random hpf with 100× magnification. Results are reported as the number of positive cells per square millimeter.A tissue section was stained with hematoxylin and eosin. Eosinophils were identified on the basis of their cellular features and distinct cytoplasmic eosinophilic granules. For all polyp samples, a serial section was stained with a tryptase mAB, a specific marker for mast cells as described previously.7Takabayashi T. Kato A. Peters A.T. Suh L.A. Carter R. Norton J. et al.Glandular mast cells with distinct phenotype are highly elevated in chronic rhinosinusitis with nasal polyps.J Allergy Clin Immunol. 2012; 130: 410-420.e5Google Scholar Cells were counted and data expressed as described above for basophils.Data are presented as mean ± SEM. Comparisons between groups were assessed by using ANOVA and Newman-Keuls multiple comparison tests. Correlations were measured using a Spearman rank correlation test. All statistical analyses were performed using GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, Calif; www.graphpad.com). A P value of less than .05 was considered statistically significant.A total of 73 sections were labeled with 2D7, including polyps from 27 patients with and without AERD (10 with AERD and 17 without AERD) and UT from 16 patients with CRSwNP, 15 patients with CRSsNP, and 15 control cases with no evidence of CRS. Cells were identified as basophils on the basis of a brown cytoplasmic granular staining pattern (Fig 1, A). Negative control slides were labeled with an isotype-matched nonimmune antibody and showed no staining.As detailed in Table I, there were no 2D7 basophils detected within the uncinate mucosa and submucosa of control subjects. A small number of these cells were detected in the UT of patients with CRSsNP, and a greater number was detected in the UT of patients with CRSwNP, though the quantities detected were not significantly greater than in control UT.Table ISummary of cell count results in different tissuesCellControlCRSsNPCRSwNPPolyp (non-AERD)Polyp (AERD)Basophils (cells per square millimeter), mean (SEM)0.35 (0.16)1.06 (0.22)3.5 (0.85)7.76 (1.71)∗P < .05 for comparison by ANOVA and Newman-Keuls multiple comparison tests when compared with the UT of patients with CRSsNP and CRSwNP. P < .001 when compared with controls.2.50 (0.86)†P < .05 for comparison by ANOVA and Newman-Keuls multiple comparison tests when compared with non-AERD polyp.Eosinophils (cells per square millimeter), mean (SEM)20.5 (15.57)14.73 (5.49)89.31 (21.49)365.1 (65.46)‡P < .05 for comparison by ANOVA and Newman-Keuls multiple comparison tests when compared with the UT of patients with CRSwNP. P < .001 when compared with the UT of patients with CRSsNP and controls.453 (45)‡P < .05 for comparison by ANOVA and Newman-Keuls multiple comparison tests when compared with the UT of patients with CRSwNP. P < .001 when compared with the UT of patients with CRSsNP and controls.Mast cells (cells per square millimeter), mean (SEM)25.21 (3.4)29.60 (0.35)∗ P < .05 for comparison by ANOVA and Newman-Keuls multiple comparison tests when compared with the UT of patients with CRSsNP and CRSwNP. P < .001 when compared with controls.† P < .05 for comparison by ANOVA and Newman-Keuls multiple comparison tests when compared with non-AERD polyp.‡ P < .05 for comparison by ANOVA and Newman-Keuls multiple comparison tests when compared with the UT of patients with CRSwNP. P < .001 when compared with the UT of patients with CRSsNP and controls. Open table in a new tab The 2D7-positive cells per square millimeter of tissue were significantly higher in the NP tissue of patients with CRSwNP than in the UT of control subjects or patients with CRSsNP and CRSwNP (Fig 1, B). This count was also significantly higher when compared with the corresponding UT of the same patients with CRSwNP, paired sets of the polyp tissue and the UT having been evaluated from 12 patients with CRSwNP. Interestingly, the number of basophils detected in NPs from subjects with AERD was not higher than in normal or CRS UT and was significantly lower than in NPs from patients without AERD.Eosinophil counts in the UT of control patients with no evidence of sinusitis were significantly lower than in the UT of patients with CRSwNP (Table I). Polyp tissue had a significantly higher number of eosinophils than did the UT of all groups, including controls and patients with CRSsNP and CRSwNP. Polyps from subjects with AERD had a significantly higher eosinophil count than did the UT of all groups, with a trend toward higher levels than in non-AERD polyps.Basophil and eosinophil counts in all tissues (excluding AERD polyps) significantly correlated with one another (P < .001; Spearman r = 0.67) (Fig 1, D). But the basophil and mast cell counts did not correlate.Basophils and eosinophils utilize similar pathways for recruitment and might be expected to be recruited to an inflammatory site in parallel.8Bochner B.S. Schleimer R.P. Mast cells, basophils, and eosinophils: distinct but overlapping pathways for recruitment.Immunol Rev. 2001; 179: 5-15Google Scholar We therefore calculated the ratio of eosinophils to basophils by dividing the total number of eosinophils per hpf by the number of basophils per hpf in a fixed area in serially cut slides. The eosinophil to basophil ratio in normal UT had a mean value of 3.0; this value was 13.1 in CRSsNP UT and 49.0 in CRSwNP UT. Furthermore, the magnitude of the ratio of eosinophils to basophils was highest in NPs, with a mean of 89.2 in non-AERD polyps and 287.3 in AERD polyps. Values of this ratio were significantly higher in polyps than in the UT of CRSwNP, CRSsNP, and control cases.The blood basophil counts within 6 months of the time of surgery were available for 40 cases: 13 with analysis of polyp tissue and 27 with analysis of the UT. There was no correlation between the blood basophil count and the number of basophils in either the polyp tissue or the UT. This appears to exclude systemic basophilia as a likely cause of the observed increase in the number of basophils in the polyp tissue. We also correlated the basophil numbers in polyps with total serum IgE (available in 13 cases) and the presence of anosmia or hyposmia as a marker for disease severity and positive skin test result. None of these comparisons was significant (both including and excluding AERD polyps). Potentially other patient-related factors, such as treatment, might have affected the number of basophils. We compared the number of basophils in the UT and the polyp tissue of patients who were on oral steroids at the time of surgery compared with those who were not (both including and excluding AERD cases); the results were not statistically significant.In this letter, we report that there is a trend for increased numbers of basophils in the UT from patients with CRS and that NP tissue in patients without AERD contains clearly elevated numbers of basophils compared with the UT from both patients and controls; polyps from patients with AERD had a trend toward elevated basophils but significantly fewer basophils than did polyps from patients without AERD. As expected on the basis of reports in the literature,9Payne S.C. Early S.B. Huyett P. Han J.K. Borish L. Steinke J.W. Evidence for distinct histologic profile of nasal polyps with and without eosinophilia.Laryngoscope. 2011; 121: 2262-2267Google Scholar eosinophils were also elevated in the UT from patients with CRS and were especially prominent in NP tissue from subjects both with and without AERD. The lower number of detected basophils in polyps from subjects with AERD raises the question as to whether these cells were never recruited to these polyp samples or could not be detected by 2D7. Basophils recruited to AERD NP tissue may be activated rapidly in vivo, may degranulate more quickly and more completely after homing to the polyps, and thus lose their granule proteins, including the protein detected by 2D7, as described previously in skin biopsies obtained during the late-phase response to allergen challenge.10Irani A.M. Huang C. Xia H.Z. Kepley C. Nafie A. Fouda E.D. et al.Immunohistochemical detection of human basophils in late-phase skin reactions.J Allergy Clin Immunol. 1998; 101: 354-362Google ScholarThe number of tissue basophils correlated with the number of tissue eosinophils, suggesting that these cells are associated in the inflammation found in CRS. Despite this significant correlation, there was a relative enrichment of eosinophils compared with basophils in polyp tissue, as the eosinophil/basophil ratio was close to 100 in polyps, a value significantly higher than that in the UT from patients with CRS. This ratio in the NP tissue is also markedly higher than ratios calculated from counts reported in the literature using other respiratory tissues; the ratio was about 10 in the bronchial mucosa in patients with asthma in one study11Macfarlane A.J. Kon O.M. Smith S.J. Zeibecoglou K. Khan L.N. Barata L.T. et al.Basophils, eosinophils, and mast cells in atopic and nonatopic asthma and in late-phase allergic reactions in the lung and skin.J Allergy Clin Immunol. 2000; 105: 99-107Google Scholar and about 20 in nasal submucosa in patients with allergic rhinitis at the peak of season in another.5Wilson D.R. Irani A.M. Walker S.M. Jacobson M.R. Mackay I.S. Schwartz L.B. et al.Grass pollen immunotherapy inhibits seasonal increases in basophils and eosinophils in the nasal epithelium.Clin Exp Allergy. 2001; 31: 1705-1713Google Scholar This significant enrichment of eosinophils in NPs compared with basophils might reflect selective recruitment of eosinophils to the polyp or sustained survival of eosinophils compared with basophils in polyp tissue and when compared to other respiratory tissues undergoing an allergic response. It is also possible that our measurements of basophils are underestimates due to degranulation as mentioned above or for another reason. For example, it is possible that recruited basophils might exit the polyp into the nasal cavity preferentially compared with eosinophils. There are multiple studies demonstrating basophils in respiratory secretions in asthma during asthmatic attacks or after allergen challenge and allergic rhinitis in the peak of allergy season.5Wilson D.R. Irani A.M. Walker S.M. Jacobson M.R. Mackay I.S. Schwartz L.B. et al.Grass pollen immunotherapy inhibits seasonal increases in basophils and eosinophils in the nasal epithelium.Clin Exp Allergy. 2001; 31: 1705-1713Google Scholar, 12Hastie R. Heroy III, J.H. Levy D.A. Basophil leukocytes and mast cells in human nasal secretions and scrapings studied by light microscopy.Lab Invest. 1979; 40: 554-561Google Scholar In one study, basophil numbers in the sputum increased by almost 200-fold from baseline values during the allergy season compared with eosinophils, which increased by approximately 30-fold.4Gauvreau G.M. Lee J.M. Watson R.M. Irani A.M. Schwartz L.B. O'Byrne P.M. Increased numbers of both airway basophils and mast cells in sputum after allergen inhalation challenge of atopic asthmatics.Am J Respir Crit Care Med. 2000; 161: 1473-1478Google Scholar This could be indicative of a higher tendency of basophils to migrate into the airway lumen.The presence of basophils in the NP tissue raises questions about their modes of activation and potential role in perpetuating or enhancing NP inflammation.The level of IL-3 mRNA, a major cytokine for development and survival of basophils, is reportedly not elevated in CRS nasal and polyp tissue,13Rudack C. Stoll W. Bachert C. Cytokines in nasal polyposis, acute and chronic sinusitis.Am J Rhinol. 1998; 12: 383-388Google Scholar a conclusion confirmed in a microarray analysis done by our group.14Seshadri 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: 920-928Google Scholar However, as supported by the presence of basophils in IL-3–deficient mice,15Lantz C.S. Boesiger J. Song C.H. Mach N. Kobayashi T. Mulligan R.C. et al.Role for interleukin-3 in mast-cell and basophil development and in immunity to parasites.Nature. 1998; 392: 90-93Google Scholar these cells can be expanded by other cytokines, such as thymic stromal lymphopoietin16Perrigoue J.G. Saenz S.A. Siracusa M.C. Allenspach E.J. Taylor B.C. Giacomin P.R. et al.MHC class II-dependent basophil-CD4+ T cell interactions promote T(H)2 cytokine-dependent immunity.Nat Immunol. 2009; 10: 697-705Google Scholar and IL-33,17Suzukawa M. Iikura M. Koketsu R. Nagase H. Tamura C. Komiya A. et al.An IL-1 cytokine member, IL-33, induces human basophil activation via its ST2 receptor.J Immunol. 2008; 181: 5981-5989Google Scholar the former of which is significantly expressed in NP epithelial cells extracted from patients with CRS.18Nagarkar D.R. Poposki J.A. Tan B.K. Comeau M.R. Peters A.T. Hulse K.E. et al.Thymic stromal lymphopoietin activity is increased in nasal polyps of patients with chronic rhinosinusitis.J Allergy Clin Immunol. 2013; 132: 593-600.e12Abstract Full Text Full Text PDF Scopus (166) Google Scholar Basophils can be activated by cross-linking of allergen-specific IgE as well as via bacterial peptidoglycans through TLR-2,2Schroeder J.T. Basophils: emerging roles in the pathogenesis of allergic disease.Immunol Rev. 2011; 242: 144-160Google Scholar or by formyl-methionyl-leucyl-phenylalanine19Siraganian R.P. Hook W.A. Mechanism of histamine release by formyl methionine-containing peptides.J Immunol. 1977; 119: 2078-2083Google Scholar or serine proteases through other receptors.2Schroeder J.T. Basophils: emerging roles in the pathogenesis of allergic disease.Immunol Rev. 2011; 242: 144-160Google Scholar Thus, bacterial infection or colonization of the sinus mucosa, commonly observed in CRS, can theoretically activate the basophils we have detected in the NP tissue.Once recruited and activated, basophils can potentially exert inflammatory effects through several mediators. Basophils are a remarkable source of vasoactive histamine and leukotrienes that can cause tissue edema, and hence contribute to nasal congestion. Improvement of nasal congestion in response to leukotriene antagonists suggests an important role for these mediators, derived from basophils and other cells, in the symptomatology of patients with CRS.Basophils can also participate in recruiting other leukocytes to sites of allergic inflammation. Basophils secrete large quantities of IL-420MacGlashan Jr., D. White J.M. Huang S.K. Ono S.J. Schroeder J.T. Lichtenstein L.M. Secretion of IL-4 from human basophils: the relationship between IL-4 mRNA and protein in resting and stimulated basophils.J Immunol. 1994; 152: 3006-3016Google Scholar and IL-13,2Schroeder J.T. Basophils: emerging roles in the pathogenesis of allergic disease.Immunol Rev. 2011; 242: 144-160Google Scholar cytokines that are known to promote goblet cell hyperplasia and mucus production in asthma. Hyperplasia of goblet cells, submucosal glands, and excessive mucus secretion are all features that are commonly seen in patients with CRS. IL-4 can also increase the expression of integrin receptors such as vascular cell adhesion molecule-1 that can bind VLA-4–expressing leukocytes including lymphocytes, monocytes, and eosinophils,21Ying S. Meng Q. Barata L.T. Robinson D.S. Durham S.R. Kay A.B. Associations between IL-13 and IL-4 (mRNA and protein), vascular cell adhesion molecule-1 expression, and the infiltration of eosinophils, macrophages, and T cells in allergen-induced late-phase cutaneous reactions in atopic subjects.J Immunol. 1997; 158: 5050-5057Google Scholar and is shown to be correlated with the presence of eosinophils in CRS.22Jahnsen F.L. Haraldsen G. Aanesen J.P. Haye R. Brandtzaeg P. Eosinophil infiltration is related to increased expression of vascular cell adhesion molecule-1 in nasal polyps.Am J Respir Cell Mol Biol. 1995; 12: 624-632Google Scholar It should thus be considered that basophils may be involved in the recruitment of other inflammatory cells to the polyp and promoting inflammation in CRS.In conclusion, we found a significantly higher number of basophils in the NP tissue than in the UT of controls and patients with CRSwNP without AERD. In an environment rich in potential activators, and considering their ability to produce multiple inflammatory mediators, basophils may thus make an important contribution to the pathogenesis and symptomatology of CRS. Chronic rhinosinusitis (CRS) is a common chronic disease with significant burden and effect on the quality of life of affected individuals, characterized by inflammation of the nasal mucosa and paranasal sinuses. Patients can be subdivided into CRS with nasal polyps (CRSwNP) and CRS without nasal polyps (CRSsNP). In addition, aspirin exacerbated respiratory disease (AERD) is a subgroup of CRSwNP characterized by the triad of CRSwNP, asthma, and hypersensitivity to aspirin or nonsteroidal antiinflammatory drugs. Eosinophilia is evident in the nasal mucosa and submucosa of nasal polyps (NPs) from patients in Europe and the United States, but less so in Asian countries such as China or Japan.1Zhang N. Van Zele T. Perez-Novo C. Van Bruaene N. Holtappels G. DeRuyck N. et al.Different types of T-effector cells orchestrate mucosal inflammation in chronic sinus disease.J Allergy Clin Immunol. 2008; 122: 961-968Google Scholar In this study we attempted, for the first time, to evaluate the role of another important inflammatory leukocyte, the basophil, in CRS. Basophils are granulocytes found mainly in the circulation and are known to have a role in allergic diseases, parasite expulsion, and immunity against ectoparasites.2Schroeder J.T. Basophils: emerging roles in the pathogenesis of allergic disease.Immunol Rev. 2011; 242: 144-160Google Scholar Basophil numbers are elevated in the bronchial mucosa and submucosa of asthmatic patients, the nasal submucosa in patients with allergic rhinitis, and in the skin of those with multiple inflammatory dermatologic conditions, including eczema and urticaria.2Schroeder J.T. Basophils: emerging roles in the pathogenesis of allergic disease.Immunol Rev. 2011; 242: 144-160Google Scholar, 3Nouri-Aria K.T. Irani A.M. Jacobson M.R. O'Brien F. Varga E.M. Till S.J. et al.Basophil recruitment and IL-4 production during human allergen-induced late asthma.J Allergy Clin Immunol. 2001; 108: 205-211Google Scholar, 4Gauvreau G.M. Lee J.M. Watson R.M. Irani A.M. Schwartz L.B. O'Byrne P.M. Increased numbers of both airway basophils and mast cells in sputum after allergen inhalation challenge of atopic asthmatics.Am J Respir Crit Care Med. 2000; 161: 1473-1478Google Scholar, 5Wilson D.R. Irani A.M. Walker S.M. Jacobson M.R. Mackay I.S. Schwartz L.B. et al.Grass pollen immunotherapy inhibits seasonal increases in basophils and eosinophils in the nasal epithelium.Clin Exp Allergy. 2001; 31: 1705-1713Google Scholar Although CRSwNP is a highly TH2-biased disease, making basophil involvement likely, there are no reports of basophils in CRS. In the current study we used 2D7, a mouse mAb against a basophil-specific intermediate form of major basic protein,6Plager D.A. Weiss E.A. Kephart G.M. Mocharla R.M. Matsumoto R. Checkel J.L. et al.Identification of basophils by a mAb directed against pro-major basic protein 1.J Allergy Clin Immunol. 2006; 117: 626-634Google Scholar to detect basophils in uncinate tissue (UT) or polyp tissue obtained during surgery from patients with CRS and control subjects as described previously.7Takabayashi T. Kato A. Peters A.T. Suh L.A. Carter R. Norton J. et al.Glandular mast cells with distinct phenotype are highly elevated in chronic rhinosinusitis with nasal polyps.J Allergy Clin Immunol. 2012; 130: 410-420.e5Google Scholar This antibody is shown to be a specific marker for basophils and does not recognize any other cell type.6Plager D.A. Weiss E.A. Kephart G.M. Mocharla R.M. Matsumoto R. Checkel J.L. et al.Identification of basophils by a mAb directed against pro-major basic protein 1.J Allergy Clin Immunol. 2006; 117: 626-634Google Scholar We used the immunohistochemistry protocol detailed in previous studies using 2D7.3Nouri-Aria K.T. Irani A.M. Jacobson M.R. O'Brien F. Varga E.M. Till S.J. et al.Basophil recruitment and IL-4 production during human allergen-induced late asthma.J Allergy Clin Immunol. 2001; 108: 205-211Google Scholar, 5Wilson D.R. Irani A.M. Walker S.M. Jacobson M.R. Mackay I.S. Schwartz L.B. et al.Grass pollen immunotherapy inhibits seasonal increases in basophils and eosinophils in the nasal epithelium.Clin Exp Allergy. 2001; 31: 1705-1713Google Scholar Mucosa and submucosa in the tissue biopsies were delineated, and the total number of positively stained cells in each field was counted in 50 random hpf with 100× magnification. Results are reported as the number of positive cells per square millimeter. A tissue section was stained with hematoxylin and eosin. Eosinophils were identified on the basis of their cellular features and distinct cytoplasmic eosinophilic granules. For all polyp samples, a serial section was stained with a tryptase mAB, a specific marker for mast cells as described previously.7Takabayashi T. Kato A. Peters A.T. Suh L.A. Carter R. Norton J. et al.Glandular mast cells with distinct phenotype are highly elevated in chronic rhinosinusitis with nasal polyps.J Allergy Clin Immunol. 2012; 130: 410-420.e5Google Scholar Cells were counted and data expressed as described above for basophils. Data are presented as mean ± SEM. Comparisons between groups were assessed by using ANOVA and Newman-Keuls multiple comparison tests. Correlations were measured using a Spearman rank correlation test. All statistical analyses were performed using GraphPad Prism version 6.00 for Windows (GraphPad Software, La Jolla, Calif; www.graphpad.com). A P value of less than .05 was considered statistically significant. A total of 73 sections were labeled with 2D7, including polyps from 27 patients with and without AERD (10 with AERD and 17 without AERD) and UT from 16 patients with CRSwNP, 15 patients with CRSsNP, and 15 control cases with no evidence of CRS. Cells were identified as basophils on the basis of a brown cytoplasmic granular staining pattern (Fig 1, A). Negative control slides were labeled with an isotype-matched nonimmune antibody and showed no staining. As detailed in Table I, there were no 2D7 basophils detected within the uncinate mucosa and submucosa of control subjects. A small number of these cells were detected in the UT of patients with CRSsNP, and a greater number was detected in the UT of patients with CRSwNP, though the quantities detected were not significantly greater than in control UT. The 2D7-positive cells per square millimeter of tissue were significantly higher in the NP tissue of patients with CRSwNP than in the UT of control subjects or patients with CRSsNP and CRSwNP (Fig 1, B). This count was also significantly higher when compared with the corresponding UT of the same patients with CRSwNP, paired sets of the polyp tissue and the UT having been evaluated from 12 patients with CRSwNP. Interestingly, the number of basophils detected in NPs from subjects with AERD was not higher than in normal or CRS UT and was significantly lower than in NPs from patients without AERD. Eosinophil counts in the UT of control patients with no evidence of sinusitis were significantly lower than in the UT of patients with CRSwNP (Table I). Polyp tissue had a significantly higher number of eosinophils than did the UT of all groups, including controls and patients with CRSsNP and CRSwNP. Polyps from subjects with AERD had a significantly higher eosinophil count than did the UT of all groups, with a trend toward higher levels than in non-AERD polyps. Basophil and eosinophil counts in all tissues (excluding AERD polyps) significantly correlated with one another (P < .001; Spearman r = 0.67) (Fig 1, D). But the basophil and mast cell counts did not correlate. Basophils and eosinophils utilize similar pathways for recruitment and might be expected to be recruited to an inflammatory site in parallel.8Bochner B.S. Schleimer R.P. Mast cells, basophils, and eosinophils: distinct but overlapping pathways for recruitment.Immunol Rev. 2001; 179: 5-15Google Scholar We therefore calculated the ratio of eosinophils to basophils by dividing the total number of eosinophils per hpf by the number of basophils per hpf in a fixed area in serially cut slides. The eosinophil to basophil ratio in normal UT had a mean value of 3.0; this value was 13.1 in CRSsNP UT and 49.0 in CRSwNP UT. Furthermore, the magnitude of the ratio of eosinophils to basophils was highest in NPs, with a mean of 89.2 in non-AERD polyps and 287.3 in AERD polyps. Values of this ratio were significantly higher in polyps than in the UT of CRSwNP, CRSsNP, and control cases. The blood basophil counts within 6 months of the time of surgery were available for 40 cases: 13 with analysis of polyp tissue and 27 with analysis of the UT. There was no correlation between the blood basophil count and the number of basophils in either the polyp tissue or the UT. This appears to exclude systemic basophilia as a likely cause of the observed increase in the number of basophils in the polyp tissue. We also correlated the basophil numbers in polyps with total serum IgE (available in 13 cases) and the presence of anosmia or hyposmia as a marker for disease severity and positive skin test result. None of these comparisons was significant (both including and excluding AERD polyps). Potentially other patient-related factors, such as treatment, might have affected the number of basophils. We compared the number of basophils in the UT and the polyp tissue of patients who were on oral steroids at the time of surgery compared with those who were not (both including and excluding AERD cases); the results were not statistically significant. In this letter, we report that there is a trend for increased numbers of basophils in the UT from patients with CRS and that NP tissue in patients without AERD contains clearly elevated numbers of basophils compared with the UT from both patients and controls; polyps from patients with AERD had a trend toward elevated basophils but significantly fewer basophils than did polyps from patients without AERD. As expected on the basis of reports in the literature,9Payne S.C. Early S.B. Huyett P. Han J.K. Borish L. Steinke J.W. Evidence for distinct histologic profile of nasal polyps with and without eosinophilia.Laryngoscope. 2011; 121: 2262-2267Google Scholar eosinophils were also elevated in the UT from patients with CRS and were especially prominent in NP tissue from subjects both with and without AERD. The lower number of detected basophils in polyps from subjects with AERD raises the question as to whether these cells were never recruited to these polyp samples or could not be detected by 2D7. Basophils recruited to AERD NP tissue may be activated rapidly in vivo, may degranulate more quickly and more completely after homing to the polyps, and thus lose their granule proteins, including the protein detected by 2D7, as described previously in skin biopsies obtained during the late-phase response to allergen challenge.10Irani A.M. Huang C. Xia H.Z. Kepley C. Nafie A. Fouda E.D. et al.Immunohistochemical detection of human basophils in late-phase skin reactions.J Allergy Clin Immunol. 1998; 101: 354-362Google Scholar The number of tissue basophils correlated with the number of tissue eosinophils, suggesting that these cells are associated in the inflammation found in CRS. Despite this significant correlation, there was a relative enrichment of eosinophils compared with basophils in polyp tissue, as the eosinophil/basophil ratio was close to 100 in polyps, a value significantly higher than that in the UT from patients with CRS. This ratio in the NP tissue is also markedly higher than ratios calculated from counts reported in the literature using other respiratory tissues; the ratio was about 10 in the bronchial mucosa in patients with asthma in one study11Macfarlane A.J. Kon O.M. Smith S.J. Zeibecoglou K. Khan L.N. Barata L.T. et al.Basophils, eosinophils, and mast cells in atopic and nonatopic asthma and in late-phase allergic reactions in the lung and skin.J Allergy Clin Immunol. 2000; 105: 99-107Google Scholar and about 20 in nasal submucosa in patients with allergic rhinitis at the peak of season in another.5Wilson D.R. Irani A.M. Walker S.M. Jacobson M.R. Mackay I.S. Schwartz L.B. et al.Grass pollen immunotherapy inhibits seasonal increases in basophils and eosinophils in the nasal epithelium.Clin Exp Allergy. 2001; 31: 1705-1713Google Scholar This significant enrichment of eosinophils in NPs compared with basophils might reflect selective recruitment of eosinophils to the polyp or sustained survival of eosinophils compared with basophils in polyp tissue and when compared to other respiratory tissues undergoing an allergic response. It is also possible that our measurements of basophils are underestimates due to degranulation as mentioned above or for another reason. For example, it is possible that recruited basophils might exit the polyp into the nasal cavity preferentially compared with eosinophils. There are multiple studies demonstrating basophils in respiratory secretions in asthma during asthmatic attacks or after allergen challenge and allergic rhinitis in the peak of allergy season.5Wilson D.R. Irani A.M. Walker S.M. Jacobson M.R. Mackay I.S. Schwartz L.B. et al.Grass pollen immunotherapy inhibits seasonal increases in basophils and eosinophils in the nasal epithelium.Clin Exp Allergy. 2001; 31: 1705-1713Google Scholar, 12Hastie R. Heroy III, J.H. Levy D.A. Basophil leukocytes and mast cells in human nasal secretions and scrapings studied by light microscopy.Lab Invest. 1979; 40: 554-561Google Scholar In one study, basophil numbers in the sputum increased by almost 200-fold from baseline values during the allergy season compared with eosinophils, which increased by approximately 30-fold.4Gauvreau G.M. Lee J.M. Watson R.M. Irani A.M. Schwartz L.B. O'Byrne P.M. Increased numbers of both airway basophils and mast cells in sputum after allergen inhalation challenge of atopic asthmatics.Am J Respir Crit Care Med. 2000; 161: 1473-1478Google Scholar This could be indicative of a higher tendency of basophils to migrate into the airway lumen. The presence of basophils in the NP tissue raises questions about their modes of activation and potential role in perpetuating or enhancing NP inflammation. The level of IL-3 mRNA, a major cytokine for development and survival of basophils, is reportedly not elevated in CRS nasal and polyp tissue,13Rudack C. Stoll W. Bachert C. Cytokines in nasal polyposis, acute and chronic sinusitis.Am J Rhinol. 1998; 12: 383-388Google Scholar a conclusion confirmed in a microarray analysis done by our group.14Seshadri 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: 920-928Google Scholar However, as supported by the presence of basophils in IL-3–deficient mice,15Lantz C.S. Boesiger J. Song C.H. Mach N. Kobayashi T. Mulligan R.C. et al.Role for interleukin-3 in mast-cell and basophil development and in immunity to parasites.Nature. 1998; 392: 90-93Google Scholar these cells can be expanded by other cytokines, such as thymic stromal lymphopoietin16Perrigoue J.G. Saenz S.A. Siracusa M.C. Allenspach E.J. Taylor B.C. Giacomin P.R. et al.MHC class II-dependent basophil-CD4+ T cell interactions promote T(H)2 cytokine-dependent immunity.Nat Immunol. 2009; 10: 697-705Google Scholar and IL-33,17Suzukawa M. Iikura M. Koketsu R. Nagase H. Tamura C. Komiya A. et al.An IL-1 cytokine member, IL-33, induces human basophil activation via its ST2 receptor.J Immunol. 2008; 181: 5981-5989Google Scholar the former of which is significantly expressed in NP epithelial cells extracted from patients with CRS.18Nagarkar D.R. Poposki J.A. Tan B.K. Comeau M.R. Peters A.T. Hulse K.E. et al.Thymic stromal lymphopoietin activity is increased in nasal polyps of patients with chronic rhinosinusitis.J Allergy Clin Immunol. 2013; 132: 593-600.e12Abstract Full Text Full Text PDF Scopus (166) Google Scholar Basophils can be activated by cross-linking of allergen-specific IgE as well as via bacterial peptidoglycans through TLR-2,2Schroeder J.T. Basophils: emerging roles in the pathogenesis of allergic disease.Immunol Rev. 2011; 242: 144-160Google Scholar or by formyl-methionyl-leucyl-phenylalanine19Siraganian R.P. Hook W.A. Mechanism of histamine release by formyl methionine-containing peptides.J Immunol. 1977; 119: 2078-2083Google Scholar or serine proteases through other receptors.2Schroeder J.T. Basophils: emerging roles in the pathogenesis of allergic disease.Immunol Rev. 2011; 242: 144-160Google Scholar Thus, bacterial infection or colonization of the sinus mucosa, commonly observed in CRS, can theoretically activate the basophils we have detected in the NP tissue. Once recruited and activated, basophils can potentially exert inflammatory effects through several mediators. Basophils are a remarkable source of vasoactive histamine and leukotrienes that can cause tissue edema, and hence contribute to nasal congestion. Improvement of nasal congestion in response to leukotriene antagonists suggests an important role for these mediators, derived from basophils and other cells, in the symptomatology of patients with CRS. Basophils can also participate in recruiting other leukocytes to sites of allergic inflammation. Basophils secrete large quantities of IL-420MacGlashan Jr., D. White J.M. Huang S.K. Ono S.J. Schroeder J.T. Lichtenstein L.M. Secretion of IL-4 from human basophils: the relationship between IL-4 mRNA and protein in resting and stimulated basophils.J Immunol. 1994; 152: 3006-3016Google Scholar and IL-13,2Schroeder J.T. Basophils: emerging roles in the pathogenesis of allergic disease.Immunol Rev. 2011; 242: 144-160Google Scholar cytokines that are known to promote goblet cell hyperplasia and mucus production in asthma. Hyperplasia of goblet cells, submucosal glands, and excessive mucus secretion are all features that are commonly seen in patients with CRS. IL-4 can also increase the expression of integrin receptors such as vascular cell adhesion molecule-1 that can bind VLA-4–expressing leukocytes including lymphocytes, monocytes, and eosinophils,21Ying S. Meng Q. Barata L.T. Robinson D.S. Durham S.R. Kay A.B. Associations between IL-13 and IL-4 (mRNA and protein), vascular cell adhesion molecule-1 expression, and the infiltration of eosinophils, macrophages, and T cells in allergen-induced late-phase cutaneous reactions in atopic subjects.J Immunol. 1997; 158: 5050-5057Google Scholar and is shown to be correlated with the presence of eosinophils in CRS.22Jahnsen F.L. Haraldsen G. Aanesen J.P. Haye R. Brandtzaeg P. Eosinophil infiltration is related to increased expression of vascular cell adhesion molecule-1 in nasal polyps.Am J Respir Cell Mol Biol. 1995; 12: 624-632Google Scholar It should thus be considered that basophils may be involved in the recruitment of other inflammatory cells to the polyp and promoting inflammation in CRS. In conclusion, we found a significantly higher number of basophils in the NP tissue than in the UT of controls and patients with CRSwNP without AERD. In an environment rich in potential activators, and considering their ability to produce multiple inflammatory mediators, basophils may thus make an important contribution to the pathogenesis and symptomatology of CRS.