Endothelial L-Selectin Ligands in Sinus Mucosa during Chronic Maxillary Rhinosinusitis
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美国呼吸和危急护理医学 2005年第6期
Department of Eye, Ear
Oral Diseases, Tampere University Hospital and University of Tampere, Tampere
Department of Otorhinolaryngology, Mikkeli Central Hospital, Mikkeli
Transplantation Laboratory, Departments of Pathology and Bacteriology and Immunology, Haartman Institute, Helsinki
Rational Drug Design Program, Biomedicum, University of Helsinki, Helsinki
Laboratory Diagnostics, Helsinki University Central Hospital, Helsinki, Finland
ABSTRACT
Rationale: Chronic rhinosinusitis is characterized by persistent inflammation of the nasal and paranasal mucosa with numerous emigrated leukocytes. L-selectin on leukocytes and its endothelial glycosylated ligands initiate organ-specific leukocyte infiltration into inflamed tissues. Objectives: The purpose of this study was to evaluate the endothelial expression of functionally active endothelial L-selectin ligands, sulfated sialyl Lewis x, in maxillary sinus mucosa from patients with chronic rhinosinusitis and from normal control subjects. Methods: Maxillary sinus mucosa specimens (116) were obtained surgically and immunohistochemically stained with monoclonal antibodies detecting sialyl Lewis x or sulfated extended core 1 lactosamines. The severity of the inflammation was determined by intraoperative endoscopic findings, computed tomography scans, and histopathologic assessment of the specimens. Measurements and Main Results: The percentage of vessels expressing endothelial sulfated sialyl Lewis x epitopes increased during chronic rhinosinusitis compared with uninflamed control tissue, especially in patients with additional allergic rhinitis, and decreased in specimens from aspirin-intolerant patients with preoperative oral corticosteroid treatment. In addition, the expression level of endothelial sulfated sialyl Lewis x epitopes and the number of mucosal eosinophils correlated with the severity of the inflammation, and decreased in specimens taken 9 months postoperatively compared with intraoperative samples, especially in patients with intranasal corticosteroid treatment. Conclusions: Our results suggest that functionally active L-selectin ligands might guide leukocyte traffic into maxillary sinus mucosa preferentially in patients with severe findings of chronic maxillary rhinosinusitis, thus leading to aggravation of the inflammation.
Key Words: adhesion molecules L-selectin maxillary rhinosinusitis sialyl Lewis x
Chronic rhinosinusitis is one of the most common chronic conditions with increasing incidence; however, there are limitations in both a plausible etiology and an optimal treatment (1, 2). It shares numerous features with both allergic rhinitis and asthma (2, 3). In the pathophysiology of both chronic rhinosinusitis and allergic rhinitis, eosinophils, helper T lymphocytes, and other leukocytes represent important components of the inflammatory response (3, 4). Allergy is a known contributing factor to chronic rhinosinusitis (4, 5). Patients with rhinosinusitis and concomitant allergic rhinitis have a higher number of eosinophils as well as activated eosinophils infiltrating the sinus mucosa compared with those having only chronic rhinosinusitis (6).
In most organs, leukocyte recruitment proceeds in a cascade-like fashion from capture to rolling to a systematic decrease in rolling velocity to firm adhesion and transmigration (7). Recruitment is initiated by the tethering and rolling of leukocytes on endothelial cells, which is mediated by selectins and their glycosylated ligands (7eC9). These interactions are followed by a chemokine-mediated activation of integrins resulting in firm adhesion and, finally, leukocyte migration across the microvascular postcapillary endothelium (10, 11). L-selectin is expressed on most leukocytes and binds to a group of glycoproteins expressed on endothelial cells of high endothelial venules in lymph nodes, on activated endothelial cells, and on other leukocytes. These glycoproteins include CD34, sulfated gp200, endomucin, glycosylation-dependent cell adhesion molecule-1, podocalyxin, endoglycan, mucosal vascular addressin cell adhesion molecule-1, and P-selectin glycoprotein ligand-1 (12eC15).
The function of L-selectin counterreceptors depends on their decoration with sialylated, sulfated, and fucosylated oligosaccharides, on the C-6 position of galactose and on the N-acetylglucosamine residues of the numerous O-glycans (16eC23). These sulfated sialyl Lewis x glycans are consistently expressed at least on the CD34 glycans of lymph node high endothelium (24). Under normal conditions, properly glycosylated L-selectin ligands are not expressed on endothelia of other than lymphatic tissues (25). However, the induction of sulfated sialyl Lewis x ligands onto the postcapillary microvascular endothelium occurs both in rodents and humans undergoing allograft rejection as well as chronic inflammatory diseases, such as asthma (25eC29). During chronic inflammation, each organ carries its own modification of sulfated sialyl Lewis x glycans, that is, ZIP code (29). In addition, enzymatically synthesized multivalent sialyl Lewis x glycans can prevent selectin-dependent lymphocyte adhesion to properly glycosylated endothelium ex vivo, pointing to a putative means to inhibit the inflammatory reaction organ selectively (25, 30).
The previous observations are expanded to study the pathogenesis of chronic maxillary rhinosinusitis. The goal of this study was to analyze whether the expression of functionally active L-selectin ligands and the number of mucosal eosinophils correlate to the severity of chronic allergic and nonallergic rhinosinusitis, and whether they are decreased by medical and operative treatment.
METHODS
Subjects
Forty-eight subjects were enrolled in this study. Characteristics of the groups of patients are shown in Table 1 (see the online supplement for additional detail about the subjects and methods). Uninflamed normal control subjects had never suffered from chronic rhinosinusitis or allergic rhinitis. Chronic maxillary rhinosinusitis was diagnosed by the presence of sinus-related symptoms for at least 12 weeks despite medical treatment, associated with abnormalities of mucosal thickening or sinus opacification on sinus computed tomography scan (31). Patients with allergic rhinitis had at least a 2-year history of allergy confirmed by a positive skin prick test reaction for at least one inhalatory allergen (32). Patients who had, in addition, aspirin sensitivity and oral corticosteroids for 5 days before surgery were studied as a separate group. The study was supported by the ethics committee of Tampere University Hospital (Tampere, Finland).
For subjects with chronic rhinosinusitis, endoscopic sinus surgery was performed under local or general anesthesia. Intra- or postoperative endoscopic findings noted were the condition of antral mucosa. Endoscopic findings were graded according to the following scale: 0 = no inflammation, 1 = mild edema, 2 = moderate edema and inflammation, 3 = severe edema and inflammation. Biopsies of the maxillary sinus mucosa were performed during operation and 9 months postoperatively. The radiologic stage of inflammation was based on opacification of the sinuses and occlusion of the osteomeatal complex on the computed tomography scans according to Lund and coworkers (33, 34). Here, radiologic Stages 0, 1, 2, and 3 indicate scores 0, 1eC3, 4eC7, and 8eC12, respectively, according to Lund and coworkers. Additional detail about the subjects and sample collection is provided in the online supplement.
Immunohistochemistry
L-selectin ligands were studied with two monoclonal antibodies (mAbs). mAb HECA-452 recognizes -2,3-sialylation and -1,3-fucosylation of lactosamine, and mAb MECA-79 recognizes an extended sulfated core 1 lactosamine structure (35eC38). mAb HECA-452 (rat IgM; 2 e/ml) and mAb MECA-79 (rat IgM; 1 e/ml) were kindly provided by S. Jalkanen (University of Turku, Turku, Finland). Anti-human CD34, Class II (mIgG1; 2 e/ml; DakoCytomation, Glostrup, Denmark), was used as a positive control for the detection of endothelial cells. mAbs 7C7 (mouse IgM; 1.2 e/ml) and TIB-146 (rat IgM; 10 e/ml), both kindly provided by S. Jalkanen, were used as negative controls. For immunohistochemical techniques and microscopic analysis of the specimens, previously described protocols were used (27eC29). Briefly, the mean number of mAb HECA-452eC and mAb MECA-79eCvessels was divided by the mean number of CD34-positive vessels from the whole specimen, yielding the percentage of sialyl Lewix x (sLex)- or sulfated sialyl Lewis x (sulfo sLex)-reactive vessels. From hemalumeCeosin-stained tissue sections, the number of eosinophils per millimeter squared was microscopically calculated and the histopathologic assessment of inflammation was based on the mucosal edema, and the quantity and composition of leukocyte infiltrate, which was semiquantitatively scored as 0 = no inflammation, 1 = mild, 2 = moderate, or 3 = severe.
Statistical Analysis
Statistics were performed with the SPSS Base 11.0 Statistical Software Package (SPSS, Chicago, IL). Data values were expressed as means ± SD. Results were analyzed first by nonparametric KruskaleCWallis one-way analysis of variance by ranks and then by nonparametric ManneCWhitney U test for multiple comparisons in different groups. The nonparametric Spearman rank correlation test was used to study the correlation between ranks of the percentage of positive vessels and the severity of inflammation. The Spearman rank-order correlation coefficient (r) was used to assess bivariate association. A two-tailed p value less than 0.05 was considered significant with all tests.
RESULTS
In maxillary sinus specimens from normal control patients, the level of endothelial sulfated sialyl Lewis x epitopes, that is, the mean percentage ± SD of mAb HECA-452eC and mAb MECA-79eCpositive vessels, was 0.2 ± 0.3 and 0.3 ± 0.4%, respectively, and the mean number ± SD of mucosal eosinophils was 18.5 ± 27.0/mm2. However, during chronic maxillary rhinosinusitis the percentage of mAb HECA-452eC and mAb MECA-79eCpositive vessels, and the number of mucosal eosinophils, were significantly increased compared with healthy control subjects (p < 0.0001 by ManneCWhitney U test; Figure 1). Moreover, the percentage of mAb MECA-79eCpositive vessels, that is, expressing the extended core 1 polylactosamine structure, was significantly increased in patients with chronic rhinosinusitis with additional allergic rhinitis compared with patients with chronic rhinosinusitis without atopy (mean percentage ± SD: 6.5 ± 5.1 and 4.6 ± 6.1%, respectively; p < 0.05 by ManneCWhitney U test) as shown in Figure 1B. Interestingly, the percentages of mAb HECA-452eC and MECA-79eCpositive vessels were significantly decreased in specimens from aspirin-sensitive patients with chronic rhinosinusitis, allergic rhinitis, and preoperative oral glucocorticoid treatment (1.5 ± 1.0 and 1.7 ± 2.0%, respectively) compared with aspirin-tolerant patients with chronic rhinosinusitis and with allergic rhinitis but without oral corticosteroid treatment (6.5 ± 5.1 and 3.9 ± 3.2%; p < 0.001 by ManneCWhitney U test) as shown in Figures 1A and 1B. All these differences were independent of the intranasal corticosteroid drug used by the patients with chronic rhinosinusitis (data nor shown).
When studying specimens taken from patients during and 9 months after functional endoscopic sinus surgery, the intraoperative number of mucosal eosinophils was independent of preoperative intranasal corticosteroid treatment (Figure 2C). Interestingly, in specimens from patients with intranasal corticosteroid treatment, the expression level of mAb HECA-452eC and mAb MECA-79eCpositive vessels (4.6 ± 3.5 and 6.6 ± 4.5%, respectively) was increased compared with patients without intranasal corticosteroids (3.0 ± 2.4% and 5.0 ± 5.9%; p < 0.05 by ManneCWhitney U test) as shown in Figures 2A and 2B. However, the expression level of mAb HECA-452eC and mAb MECA-79eCpositive vessels and the number of mucosal eosinophils were decreased significantly in 9-month follow-up specimens, especially in patients with postoperative intranasal corticosteroid treatment (1.5 ± 0.9%, 1.0 ± 1.0%, and 43 ± 47 cells/mm2, respectively) compared with the patients without postoperative intranasal corticosteroid treatment (1.8 ± 2.3%, 1.4 ± 1.7%, and 135 ± 188 cells/mm2, respectively; p < 0.01 by ManneCWhitney U test) as shown in Figures 2 and 3. All these differences were independent of atopic status of the patients (data not shown).
The expression levels of endothelial sulfated sLex glycans, as well as the number of mucosal eosinophils in intraoperative biopsies, correlated significantly with the severity of inflammation determined by three different methods: histologic score, endoscopic grade, and radiologic stage. There was already a significant correlation between the number of mucosal eosinophils as well as the percentages of mAb HECA-452eC and mAb MECA-79eCpositive vessels, and radiologic stage and endoscopic grade of the chronic maxillary rhinosinusitis (all had a p value of less than 0.01, and a correlation coefficient value r ranging from 0.6 to 0.7, Spearman rank correlation test). However, the correlation was strongest between mucosal eosinophils, mAb HECA-452eC or mAb MECA-79eCpositive vessels, and histologic score (p < 0.01, r = 0.7; p < 0.01, r = 0.8; and p < 0.01, r = 0.8 by Spearman rank correlation test, respectively) as shown in Figure 4. In addition, the percentages of mAb HECA-452eC and mAb MECA-79eCpositive vessels correlated with the number of mucosal eosinophils in intraoperative biopsies from patients with chronic maxillary rhinosinusitis (p < 0.05, r = 0.5 and p < 0.01, r = 0.7 by Spearman rank correlation test, respectively) as shown in Figure 5.
The number of mucosal eosinophils and the expression level of activated endothelial cells endothelial (sulfated sialyl Lewis x epitope) in intraoperative maxillary sinus biopsies was independent of antihistamine treatment, asthma, or nasal polyposis status of the patients with chronic rhinosinusitis (data not shown). Interestingly, the percentage of mAb MECA-79eCpositive vessels was significantly increased in intraoperative maxillary sinus mucosa from aspirin-tolerant patients with chronic rhinosinusitis with additional allergic rhinitis and nasal polyposis compared with aspirin-insensitive patients with chronic rhinosinusitis with allergic rhinitis but without nasal polyposis (mean percentages ± SD: 10.0 ± 5.4 and 5.7 ± 4.7%, respectively; p < 0.05 by ManneCWhitney U test) (data not shown). This was independent of intranasal corticosteroid treatment (data not shown).
DISCUSSION
The present study with 116 biopsies from maxillary sinus mucosa showed that normal control samples did not essentially express endothelial sulfated sialyl Lewis x glycans when detected by mAbs HECA-452 and MECA-79. However, the endothelial expression of sulfated sialyl Lewis x oligosaccharides as well as the number of eosinophils were enhanced in mucosa during chronic maxillary rhinosinusitis. This is in line with our previous findings that specific modifications of endothelial sulfated sialyl Lewis x decorations were induced de novo in bronchial mucosa during bronchial asthma as well as in several other human tissues during chronic inflammation (28, 29).
Previously, we have found that during chronic inflammation, each organ carries its own modification of sulfated sialyl Lewis x glycans, that is, ZIP code, thus providing a possible means for organ-selective leukocyte traffic (29). Here, the percentages of mAb HECA-452eC and mAb MECA-79eCpositive vessels, that is, the ZIP codes, in respiratory mucosa of maxillary sinus during chronic rhinosinusitis are similar to the ZIP codes in bronchial mucosa during asthma, and, different from other chronic inflammatory diseases. Thus the same pattern of endothelial sulfated sialyl Lewis x glycans might guide leukocytes to the respiratory mucosa of maxillary sinuses and bronchi. This confirms previous postulations that there are similarities in the pathophysiology of upper and lower airways (39, 40), and that chronic rhinosinusitis or unstable allergic rhinitis might lead to aggravation of asthma (41eC44).
Studies demonstrate that mouse and human L-selectin ligand sulfotransferases are capable of forming 6-sulfo sialyl Lewis x on core 2-branched O-glycans and, together with core 1 extension enzyme it forms the MECA-79 epitope, defined as Gal1eC4 (sulfo-6)GlcNAc1eC3Gal1eC3GalNAc1-R, which is a partial structure of 6-sulfo sialyl Lewis x on extended core 1 O-glycans (26, 45eC48). The MECA-79 antibody also binds to 6-sulfo sialyl Lewis x on extended core 1 O-glycans and inhibits both in vivo and ex vivo lymphocyte attachment to high endothelial venules by neutralizing L-selectin ligands (37, 48, 49).
According to our results, the number of mucosal eosinophils and the expression of sulfated sialyl Lewis x glycans correlated with the severity of chronic maxillary rhinosinusitis, when determined by intraoperative endoscopic findings, computed tomography scans, and histopathologic assessment of specimens. These findings are comparable to our previous findings that the expression of sulfated sialyl Lewis x glycans correlated with the severity of acute rejection of heart allografts (26). Four major pathophysiological processes are supposed to be responsible for chronic rhinosinusitis: (1) rarely, a chronic infectious disorder with prominent hyperplasia of immune cells; (2) chronic eosinophilic rhinosinusitis; (3) allergic fungal rhinosinusitis, which is a severe variant of chronic eosinophilic rhinosinusitis with fungal colonization within the sinus cavities; and (4) a chronic inflammatory disorder characterized by a mononuclear cell infiltrate and primarily mucus gland hyperplasia but with few eosinophils (5). In this study, expression of endothelial sulfated sialyl Lewis x decorations correlated with the number of mucosal eosinophils in intraoperative biopsies from patients with atopic or nonatopic chronic maxillary rhinosinusitis. Although the prevalence of mucosal eosinophils was not systematically analyzed, they seemed to be situated in the vicinity of mAb HECAeC and MECA-positive vessels (Figure 3). Delmotte and coworkers have shown that tumor necrosis factor-, a proinflammatory cytokine, is responsible for the biosynthesis of sulfated sialyl Lewis x epitope in cultured human bronchial mucosa (50). Thus, cytokines might account for the increase in endothelial sulfated sialyl Lewis x decorations, which might guide leukocyte traffic to the maxillary sinus mucosa preferentially during chronic rhinosinusitis.
Among our patients, the percentage of vessels reacting positively with the antibody recognizing the extended core 1 polylactosamine structure, mAb MECA-79, was significantly increased in patients with chronic rhinosinusitis and additional allergic rhinitis compared with those with chronic rhinosinusitis but without allergy. This might reflect the fact that the pathogenesis of chronic allergic rhinosinusitis might differ from that of nonallergic chronic rhinosinusitis. One of the possible mechanisms proposed to explain the interaction between allergic rhinitis and rhinosinusitis is that allergic rhinitis causes priming and upregulation of adhesion molecules of circulating leukocytes, making them more likely to migrate to sites of ongoing inflammation such as those caused by bacterial or viral rhinosinusitis (51). Ogata and coworkers found increased number of mucosal activated eosinophils in subjects with allergic chronic rhinosinusitis compared with those with nonallergic chronic rhinosinusitis (52). Moreover, the prevalence of eosinophilic rhinosinusitis is increased compared with noneosinophilic rhinosinusitis in patients with allergic rhinitis (5). In our study, there was an insignificant increase in the number of eosinophils in atopic patients with chronic maxillary rhinosinusitis compared with nonatopic patients with chronic maxillary rhinosinusitis.
Interestingly, among our subjects with aspirin sensitivity, allergic rhinitis, and chronic rhinosinusitis, preoperative oral glucocorticoid treatment was associated with decreased expression of sulfated sialyl Lewis x-bearing glycoforms, whereas there was an insignificant decrease in the number of mucosal eosinophils compared with atopic patients who did not have aspirin sensitivity and oral glucocorticoid treatment. Although the comparison between aspirin-intolerant subjects with or without oral glucocorticoid treatment was not done, it can be postulated that the susceptibility of eosinophils to glucocorticoids (53) might be partly due to the inhibitory effect of glucocorticoid on L-selectineCmediated migration. In patients with acute conjunctival inflammation after cataract surgery, we showed similarly that preoperative intravenous hydrocortisone inhibited leukocyte rolling and trafficking detected by an in vivo reflected-light confocal microscopy technique (54, 55). However, in the present study, patients using preoperative intranasal glucocorticoid had an increased expression level of sulfated sialyl Lewis x glycans compared with those without intranasal glucocorticoid treatment, possibly because of the heterogeneity of the treatment given or because of the insufficient penetration of intranasal glucocorticoid to maxillary sinuses before surgical enlargement of the ostia. Nine months after sinus surgery, a significant decrease both in the expression level of mAb HECAeC and MECA-positive vessels and the number of mucosal eosinophils was found preferentially in patients with intranasal corticosteroid treatment, despite the small number of postoperative biopsies. This might be partly due to the increased penetration of intranasal corticosteroids to maxillary sinuses.
Others have shown that endothelial P-selectin was strongly expressed in nasal polyp tissue and that treatment with intranasal corticosteroids reduced it (56, 57). Monoclonal antibody against L-selectin inhibited significantly in vitro T cell adhesion to nasal polyp endothelium (58), and budesonide treatment led to a significantly decreased sialyl Lewis x-binding site concentration in nasal polyp cultures (59). In the present study, the percentage of mAb MECA-79eCpositive vessels was significantly increased in maxillary sinus mucosa from atopic patients with chronic rhinosinusitis, and was even higher in patients with nasal polyposis compared with atopic patients with chronic rhinosinusitis but without diagnosed nasal polyposis. Subjects with aspirin intolerance, nasal polyps, and asthma are more likely to have eosinophilic chronic rhinosinusitis than a noneosinophilic disorder (5). Therefore, further studies about the role of L-selectin ligands in the pathogenesis of eosinophilic chronic rhinosinusitis, especially in patients with these associated diseases, need to be performed.
Taken together, the de novo expression of endothelial sulfated sialyl Lewis x glycans in maxillary sinus mucosa might provide a means for organ-selective inhibition of leukocyte trafficking. Moreover, expression of these glycans correlated with the severity and extent of sinus disease present on computed tomography imaging, endoscopic grading, and histologic grading, thus suggesting that they may be important in the pathogenesis of chronic rhinosinusitis.
Acknowledgments
The authors thank Raija Hukkila, Marja-Leena Koskinen, Eva Sutinen, and Eriika Wasenius for excellent technical assistance, and Esko Toppila for expertise in statistics.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjounals.org
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Oral Diseases, Tampere University Hospital and University of Tampere, Tampere
Department of Otorhinolaryngology, Mikkeli Central Hospital, Mikkeli
Transplantation Laboratory, Departments of Pathology and Bacteriology and Immunology, Haartman Institute, Helsinki
Rational Drug Design Program, Biomedicum, University of Helsinki, Helsinki
Laboratory Diagnostics, Helsinki University Central Hospital, Helsinki, Finland
ABSTRACT
Rationale: Chronic rhinosinusitis is characterized by persistent inflammation of the nasal and paranasal mucosa with numerous emigrated leukocytes. L-selectin on leukocytes and its endothelial glycosylated ligands initiate organ-specific leukocyte infiltration into inflamed tissues. Objectives: The purpose of this study was to evaluate the endothelial expression of functionally active endothelial L-selectin ligands, sulfated sialyl Lewis x, in maxillary sinus mucosa from patients with chronic rhinosinusitis and from normal control subjects. Methods: Maxillary sinus mucosa specimens (116) were obtained surgically and immunohistochemically stained with monoclonal antibodies detecting sialyl Lewis x or sulfated extended core 1 lactosamines. The severity of the inflammation was determined by intraoperative endoscopic findings, computed tomography scans, and histopathologic assessment of the specimens. Measurements and Main Results: The percentage of vessels expressing endothelial sulfated sialyl Lewis x epitopes increased during chronic rhinosinusitis compared with uninflamed control tissue, especially in patients with additional allergic rhinitis, and decreased in specimens from aspirin-intolerant patients with preoperative oral corticosteroid treatment. In addition, the expression level of endothelial sulfated sialyl Lewis x epitopes and the number of mucosal eosinophils correlated with the severity of the inflammation, and decreased in specimens taken 9 months postoperatively compared with intraoperative samples, especially in patients with intranasal corticosteroid treatment. Conclusions: Our results suggest that functionally active L-selectin ligands might guide leukocyte traffic into maxillary sinus mucosa preferentially in patients with severe findings of chronic maxillary rhinosinusitis, thus leading to aggravation of the inflammation.
Key Words: adhesion molecules L-selectin maxillary rhinosinusitis sialyl Lewis x
Chronic rhinosinusitis is one of the most common chronic conditions with increasing incidence; however, there are limitations in both a plausible etiology and an optimal treatment (1, 2). It shares numerous features with both allergic rhinitis and asthma (2, 3). In the pathophysiology of both chronic rhinosinusitis and allergic rhinitis, eosinophils, helper T lymphocytes, and other leukocytes represent important components of the inflammatory response (3, 4). Allergy is a known contributing factor to chronic rhinosinusitis (4, 5). Patients with rhinosinusitis and concomitant allergic rhinitis have a higher number of eosinophils as well as activated eosinophils infiltrating the sinus mucosa compared with those having only chronic rhinosinusitis (6).
In most organs, leukocyte recruitment proceeds in a cascade-like fashion from capture to rolling to a systematic decrease in rolling velocity to firm adhesion and transmigration (7). Recruitment is initiated by the tethering and rolling of leukocytes on endothelial cells, which is mediated by selectins and their glycosylated ligands (7eC9). These interactions are followed by a chemokine-mediated activation of integrins resulting in firm adhesion and, finally, leukocyte migration across the microvascular postcapillary endothelium (10, 11). L-selectin is expressed on most leukocytes and binds to a group of glycoproteins expressed on endothelial cells of high endothelial venules in lymph nodes, on activated endothelial cells, and on other leukocytes. These glycoproteins include CD34, sulfated gp200, endomucin, glycosylation-dependent cell adhesion molecule-1, podocalyxin, endoglycan, mucosal vascular addressin cell adhesion molecule-1, and P-selectin glycoprotein ligand-1 (12eC15).
The function of L-selectin counterreceptors depends on their decoration with sialylated, sulfated, and fucosylated oligosaccharides, on the C-6 position of galactose and on the N-acetylglucosamine residues of the numerous O-glycans (16eC23). These sulfated sialyl Lewis x glycans are consistently expressed at least on the CD34 glycans of lymph node high endothelium (24). Under normal conditions, properly glycosylated L-selectin ligands are not expressed on endothelia of other than lymphatic tissues (25). However, the induction of sulfated sialyl Lewis x ligands onto the postcapillary microvascular endothelium occurs both in rodents and humans undergoing allograft rejection as well as chronic inflammatory diseases, such as asthma (25eC29). During chronic inflammation, each organ carries its own modification of sulfated sialyl Lewis x glycans, that is, ZIP code (29). In addition, enzymatically synthesized multivalent sialyl Lewis x glycans can prevent selectin-dependent lymphocyte adhesion to properly glycosylated endothelium ex vivo, pointing to a putative means to inhibit the inflammatory reaction organ selectively (25, 30).
The previous observations are expanded to study the pathogenesis of chronic maxillary rhinosinusitis. The goal of this study was to analyze whether the expression of functionally active L-selectin ligands and the number of mucosal eosinophils correlate to the severity of chronic allergic and nonallergic rhinosinusitis, and whether they are decreased by medical and operative treatment.
METHODS
Subjects
Forty-eight subjects were enrolled in this study. Characteristics of the groups of patients are shown in Table 1 (see the online supplement for additional detail about the subjects and methods). Uninflamed normal control subjects had never suffered from chronic rhinosinusitis or allergic rhinitis. Chronic maxillary rhinosinusitis was diagnosed by the presence of sinus-related symptoms for at least 12 weeks despite medical treatment, associated with abnormalities of mucosal thickening or sinus opacification on sinus computed tomography scan (31). Patients with allergic rhinitis had at least a 2-year history of allergy confirmed by a positive skin prick test reaction for at least one inhalatory allergen (32). Patients who had, in addition, aspirin sensitivity and oral corticosteroids for 5 days before surgery were studied as a separate group. The study was supported by the ethics committee of Tampere University Hospital (Tampere, Finland).
For subjects with chronic rhinosinusitis, endoscopic sinus surgery was performed under local or general anesthesia. Intra- or postoperative endoscopic findings noted were the condition of antral mucosa. Endoscopic findings were graded according to the following scale: 0 = no inflammation, 1 = mild edema, 2 = moderate edema and inflammation, 3 = severe edema and inflammation. Biopsies of the maxillary sinus mucosa were performed during operation and 9 months postoperatively. The radiologic stage of inflammation was based on opacification of the sinuses and occlusion of the osteomeatal complex on the computed tomography scans according to Lund and coworkers (33, 34). Here, radiologic Stages 0, 1, 2, and 3 indicate scores 0, 1eC3, 4eC7, and 8eC12, respectively, according to Lund and coworkers. Additional detail about the subjects and sample collection is provided in the online supplement.
Immunohistochemistry
L-selectin ligands were studied with two monoclonal antibodies (mAbs). mAb HECA-452 recognizes -2,3-sialylation and -1,3-fucosylation of lactosamine, and mAb MECA-79 recognizes an extended sulfated core 1 lactosamine structure (35eC38). mAb HECA-452 (rat IgM; 2 e/ml) and mAb MECA-79 (rat IgM; 1 e/ml) were kindly provided by S. Jalkanen (University of Turku, Turku, Finland). Anti-human CD34, Class II (mIgG1; 2 e/ml; DakoCytomation, Glostrup, Denmark), was used as a positive control for the detection of endothelial cells. mAbs 7C7 (mouse IgM; 1.2 e/ml) and TIB-146 (rat IgM; 10 e/ml), both kindly provided by S. Jalkanen, were used as negative controls. For immunohistochemical techniques and microscopic analysis of the specimens, previously described protocols were used (27eC29). Briefly, the mean number of mAb HECA-452eC and mAb MECA-79eCvessels was divided by the mean number of CD34-positive vessels from the whole specimen, yielding the percentage of sialyl Lewix x (sLex)- or sulfated sialyl Lewis x (sulfo sLex)-reactive vessels. From hemalumeCeosin-stained tissue sections, the number of eosinophils per millimeter squared was microscopically calculated and the histopathologic assessment of inflammation was based on the mucosal edema, and the quantity and composition of leukocyte infiltrate, which was semiquantitatively scored as 0 = no inflammation, 1 = mild, 2 = moderate, or 3 = severe.
Statistical Analysis
Statistics were performed with the SPSS Base 11.0 Statistical Software Package (SPSS, Chicago, IL). Data values were expressed as means ± SD. Results were analyzed first by nonparametric KruskaleCWallis one-way analysis of variance by ranks and then by nonparametric ManneCWhitney U test for multiple comparisons in different groups. The nonparametric Spearman rank correlation test was used to study the correlation between ranks of the percentage of positive vessels and the severity of inflammation. The Spearman rank-order correlation coefficient (r) was used to assess bivariate association. A two-tailed p value less than 0.05 was considered significant with all tests.
RESULTS
In maxillary sinus specimens from normal control patients, the level of endothelial sulfated sialyl Lewis x epitopes, that is, the mean percentage ± SD of mAb HECA-452eC and mAb MECA-79eCpositive vessels, was 0.2 ± 0.3 and 0.3 ± 0.4%, respectively, and the mean number ± SD of mucosal eosinophils was 18.5 ± 27.0/mm2. However, during chronic maxillary rhinosinusitis the percentage of mAb HECA-452eC and mAb MECA-79eCpositive vessels, and the number of mucosal eosinophils, were significantly increased compared with healthy control subjects (p < 0.0001 by ManneCWhitney U test; Figure 1). Moreover, the percentage of mAb MECA-79eCpositive vessels, that is, expressing the extended core 1 polylactosamine structure, was significantly increased in patients with chronic rhinosinusitis with additional allergic rhinitis compared with patients with chronic rhinosinusitis without atopy (mean percentage ± SD: 6.5 ± 5.1 and 4.6 ± 6.1%, respectively; p < 0.05 by ManneCWhitney U test) as shown in Figure 1B. Interestingly, the percentages of mAb HECA-452eC and MECA-79eCpositive vessels were significantly decreased in specimens from aspirin-sensitive patients with chronic rhinosinusitis, allergic rhinitis, and preoperative oral glucocorticoid treatment (1.5 ± 1.0 and 1.7 ± 2.0%, respectively) compared with aspirin-tolerant patients with chronic rhinosinusitis and with allergic rhinitis but without oral corticosteroid treatment (6.5 ± 5.1 and 3.9 ± 3.2%; p < 0.001 by ManneCWhitney U test) as shown in Figures 1A and 1B. All these differences were independent of the intranasal corticosteroid drug used by the patients with chronic rhinosinusitis (data nor shown).
When studying specimens taken from patients during and 9 months after functional endoscopic sinus surgery, the intraoperative number of mucosal eosinophils was independent of preoperative intranasal corticosteroid treatment (Figure 2C). Interestingly, in specimens from patients with intranasal corticosteroid treatment, the expression level of mAb HECA-452eC and mAb MECA-79eCpositive vessels (4.6 ± 3.5 and 6.6 ± 4.5%, respectively) was increased compared with patients without intranasal corticosteroids (3.0 ± 2.4% and 5.0 ± 5.9%; p < 0.05 by ManneCWhitney U test) as shown in Figures 2A and 2B. However, the expression level of mAb HECA-452eC and mAb MECA-79eCpositive vessels and the number of mucosal eosinophils were decreased significantly in 9-month follow-up specimens, especially in patients with postoperative intranasal corticosteroid treatment (1.5 ± 0.9%, 1.0 ± 1.0%, and 43 ± 47 cells/mm2, respectively) compared with the patients without postoperative intranasal corticosteroid treatment (1.8 ± 2.3%, 1.4 ± 1.7%, and 135 ± 188 cells/mm2, respectively; p < 0.01 by ManneCWhitney U test) as shown in Figures 2 and 3. All these differences were independent of atopic status of the patients (data not shown).
The expression levels of endothelial sulfated sLex glycans, as well as the number of mucosal eosinophils in intraoperative biopsies, correlated significantly with the severity of inflammation determined by three different methods: histologic score, endoscopic grade, and radiologic stage. There was already a significant correlation between the number of mucosal eosinophils as well as the percentages of mAb HECA-452eC and mAb MECA-79eCpositive vessels, and radiologic stage and endoscopic grade of the chronic maxillary rhinosinusitis (all had a p value of less than 0.01, and a correlation coefficient value r ranging from 0.6 to 0.7, Spearman rank correlation test). However, the correlation was strongest between mucosal eosinophils, mAb HECA-452eC or mAb MECA-79eCpositive vessels, and histologic score (p < 0.01, r = 0.7; p < 0.01, r = 0.8; and p < 0.01, r = 0.8 by Spearman rank correlation test, respectively) as shown in Figure 4. In addition, the percentages of mAb HECA-452eC and mAb MECA-79eCpositive vessels correlated with the number of mucosal eosinophils in intraoperative biopsies from patients with chronic maxillary rhinosinusitis (p < 0.05, r = 0.5 and p < 0.01, r = 0.7 by Spearman rank correlation test, respectively) as shown in Figure 5.
The number of mucosal eosinophils and the expression level of activated endothelial cells endothelial (sulfated sialyl Lewis x epitope) in intraoperative maxillary sinus biopsies was independent of antihistamine treatment, asthma, or nasal polyposis status of the patients with chronic rhinosinusitis (data not shown). Interestingly, the percentage of mAb MECA-79eCpositive vessels was significantly increased in intraoperative maxillary sinus mucosa from aspirin-tolerant patients with chronic rhinosinusitis with additional allergic rhinitis and nasal polyposis compared with aspirin-insensitive patients with chronic rhinosinusitis with allergic rhinitis but without nasal polyposis (mean percentages ± SD: 10.0 ± 5.4 and 5.7 ± 4.7%, respectively; p < 0.05 by ManneCWhitney U test) (data not shown). This was independent of intranasal corticosteroid treatment (data not shown).
DISCUSSION
The present study with 116 biopsies from maxillary sinus mucosa showed that normal control samples did not essentially express endothelial sulfated sialyl Lewis x glycans when detected by mAbs HECA-452 and MECA-79. However, the endothelial expression of sulfated sialyl Lewis x oligosaccharides as well as the number of eosinophils were enhanced in mucosa during chronic maxillary rhinosinusitis. This is in line with our previous findings that specific modifications of endothelial sulfated sialyl Lewis x decorations were induced de novo in bronchial mucosa during bronchial asthma as well as in several other human tissues during chronic inflammation (28, 29).
Previously, we have found that during chronic inflammation, each organ carries its own modification of sulfated sialyl Lewis x glycans, that is, ZIP code, thus providing a possible means for organ-selective leukocyte traffic (29). Here, the percentages of mAb HECA-452eC and mAb MECA-79eCpositive vessels, that is, the ZIP codes, in respiratory mucosa of maxillary sinus during chronic rhinosinusitis are similar to the ZIP codes in bronchial mucosa during asthma, and, different from other chronic inflammatory diseases. Thus the same pattern of endothelial sulfated sialyl Lewis x glycans might guide leukocytes to the respiratory mucosa of maxillary sinuses and bronchi. This confirms previous postulations that there are similarities in the pathophysiology of upper and lower airways (39, 40), and that chronic rhinosinusitis or unstable allergic rhinitis might lead to aggravation of asthma (41eC44).
Studies demonstrate that mouse and human L-selectin ligand sulfotransferases are capable of forming 6-sulfo sialyl Lewis x on core 2-branched O-glycans and, together with core 1 extension enzyme it forms the MECA-79 epitope, defined as Gal1eC4 (sulfo-6)GlcNAc1eC3Gal1eC3GalNAc1-R, which is a partial structure of 6-sulfo sialyl Lewis x on extended core 1 O-glycans (26, 45eC48). The MECA-79 antibody also binds to 6-sulfo sialyl Lewis x on extended core 1 O-glycans and inhibits both in vivo and ex vivo lymphocyte attachment to high endothelial venules by neutralizing L-selectin ligands (37, 48, 49).
According to our results, the number of mucosal eosinophils and the expression of sulfated sialyl Lewis x glycans correlated with the severity of chronic maxillary rhinosinusitis, when determined by intraoperative endoscopic findings, computed tomography scans, and histopathologic assessment of specimens. These findings are comparable to our previous findings that the expression of sulfated sialyl Lewis x glycans correlated with the severity of acute rejection of heart allografts (26). Four major pathophysiological processes are supposed to be responsible for chronic rhinosinusitis: (1) rarely, a chronic infectious disorder with prominent hyperplasia of immune cells; (2) chronic eosinophilic rhinosinusitis; (3) allergic fungal rhinosinusitis, which is a severe variant of chronic eosinophilic rhinosinusitis with fungal colonization within the sinus cavities; and (4) a chronic inflammatory disorder characterized by a mononuclear cell infiltrate and primarily mucus gland hyperplasia but with few eosinophils (5). In this study, expression of endothelial sulfated sialyl Lewis x decorations correlated with the number of mucosal eosinophils in intraoperative biopsies from patients with atopic or nonatopic chronic maxillary rhinosinusitis. Although the prevalence of mucosal eosinophils was not systematically analyzed, they seemed to be situated in the vicinity of mAb HECAeC and MECA-positive vessels (Figure 3). Delmotte and coworkers have shown that tumor necrosis factor-, a proinflammatory cytokine, is responsible for the biosynthesis of sulfated sialyl Lewis x epitope in cultured human bronchial mucosa (50). Thus, cytokines might account for the increase in endothelial sulfated sialyl Lewis x decorations, which might guide leukocyte traffic to the maxillary sinus mucosa preferentially during chronic rhinosinusitis.
Among our patients, the percentage of vessels reacting positively with the antibody recognizing the extended core 1 polylactosamine structure, mAb MECA-79, was significantly increased in patients with chronic rhinosinusitis and additional allergic rhinitis compared with those with chronic rhinosinusitis but without allergy. This might reflect the fact that the pathogenesis of chronic allergic rhinosinusitis might differ from that of nonallergic chronic rhinosinusitis. One of the possible mechanisms proposed to explain the interaction between allergic rhinitis and rhinosinusitis is that allergic rhinitis causes priming and upregulation of adhesion molecules of circulating leukocytes, making them more likely to migrate to sites of ongoing inflammation such as those caused by bacterial or viral rhinosinusitis (51). Ogata and coworkers found increased number of mucosal activated eosinophils in subjects with allergic chronic rhinosinusitis compared with those with nonallergic chronic rhinosinusitis (52). Moreover, the prevalence of eosinophilic rhinosinusitis is increased compared with noneosinophilic rhinosinusitis in patients with allergic rhinitis (5). In our study, there was an insignificant increase in the number of eosinophils in atopic patients with chronic maxillary rhinosinusitis compared with nonatopic patients with chronic maxillary rhinosinusitis.
Interestingly, among our subjects with aspirin sensitivity, allergic rhinitis, and chronic rhinosinusitis, preoperative oral glucocorticoid treatment was associated with decreased expression of sulfated sialyl Lewis x-bearing glycoforms, whereas there was an insignificant decrease in the number of mucosal eosinophils compared with atopic patients who did not have aspirin sensitivity and oral glucocorticoid treatment. Although the comparison between aspirin-intolerant subjects with or without oral glucocorticoid treatment was not done, it can be postulated that the susceptibility of eosinophils to glucocorticoids (53) might be partly due to the inhibitory effect of glucocorticoid on L-selectineCmediated migration. In patients with acute conjunctival inflammation after cataract surgery, we showed similarly that preoperative intravenous hydrocortisone inhibited leukocyte rolling and trafficking detected by an in vivo reflected-light confocal microscopy technique (54, 55). However, in the present study, patients using preoperative intranasal glucocorticoid had an increased expression level of sulfated sialyl Lewis x glycans compared with those without intranasal glucocorticoid treatment, possibly because of the heterogeneity of the treatment given or because of the insufficient penetration of intranasal glucocorticoid to maxillary sinuses before surgical enlargement of the ostia. Nine months after sinus surgery, a significant decrease both in the expression level of mAb HECAeC and MECA-positive vessels and the number of mucosal eosinophils was found preferentially in patients with intranasal corticosteroid treatment, despite the small number of postoperative biopsies. This might be partly due to the increased penetration of intranasal corticosteroids to maxillary sinuses.
Others have shown that endothelial P-selectin was strongly expressed in nasal polyp tissue and that treatment with intranasal corticosteroids reduced it (56, 57). Monoclonal antibody against L-selectin inhibited significantly in vitro T cell adhesion to nasal polyp endothelium (58), and budesonide treatment led to a significantly decreased sialyl Lewis x-binding site concentration in nasal polyp cultures (59). In the present study, the percentage of mAb MECA-79eCpositive vessels was significantly increased in maxillary sinus mucosa from atopic patients with chronic rhinosinusitis, and was even higher in patients with nasal polyposis compared with atopic patients with chronic rhinosinusitis but without diagnosed nasal polyposis. Subjects with aspirin intolerance, nasal polyps, and asthma are more likely to have eosinophilic chronic rhinosinusitis than a noneosinophilic disorder (5). Therefore, further studies about the role of L-selectin ligands in the pathogenesis of eosinophilic chronic rhinosinusitis, especially in patients with these associated diseases, need to be performed.
Taken together, the de novo expression of endothelial sulfated sialyl Lewis x glycans in maxillary sinus mucosa might provide a means for organ-selective inhibition of leukocyte trafficking. Moreover, expression of these glycans correlated with the severity and extent of sinus disease present on computed tomography imaging, endoscopic grading, and histologic grading, thus suggesting that they may be important in the pathogenesis of chronic rhinosinusitis.
Acknowledgments
The authors thank Raija Hukkila, Marja-Leena Koskinen, Eva Sutinen, and Eriika Wasenius for excellent technical assistance, and Esko Toppila for expertise in statistics.
This article has an online supplement, which is accessible from this issue's table of contents at www.atsjounals.org
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