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Original Article |

Expression of Cyclooxygenase and Lipoxygenase Enzymes in Sinonasal Mucosa of Patients With Cystic Fibrosis FREE

Jonathan M. Owens, MD; Kenneth R. Shroyer, MD, PhD; Todd T. Kingdom, MD
[+] Author Affiliations

Author Affiliations: Department of Otolaryngology–Head and Neck Surgery, University of Colorado, Denver (Drs Owens and Kingdom); and Department of Pathology, Stony Brook University School of Medicine, New York, New York (Dr Shroyer). Dr Owens is now in private practice in Napa, California.


Arch Otolaryngol Head Neck Surg. 2008;134(8):825-831. doi:10.1001/archotol.134.8.825.
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Published online

Objective  To evaluate the expression of cyclooxygenase (COX) and lipoxygenase (LO) enzymes in the sinonasal mucosa of patients with cystic fibrosis (CF).

Design  Immunohistochemical staining of archived tissue.

Participants  Specimens from 9 patients with CF were analyzed; control specimens were obtained from 4 patients without a history of CF or rhinosinusitis.

Interventions  Expression of the enzymes COX-1, COX-2, 5-LO, 12-LO, and 15-LO was evaluated with the use of immunohistochemical techniques in archived sinonasal mucosal tissue from patients with CF. These results were compared with those of the control group.

Results  We noted the characteristic staining patterns of epithelium and submucosal glands for each enzyme. Statistically significant (P < .05) differences between control and CF specimens were noted in the staining intensity of columnar epithelium for COX-2 (cytoplasm) and 12-LO (cytoplasm and nucleus) and of submucosal glands for COX-2 (cytoplasm) and 12-LO (cytoplasm). No significant differences were noted for the staining intensity of COX-1, 5-LO, or 15-LO between the groups.

Conclusions  Significant differences in sinonasal mucosal expression of COX-2 and 12-LO enzymes exist between patients with CF and controls. This suggests a difference in arachidonic acid metabolism between these 2 groups.

Figures in this Article

It has been well established that patients with cystic fibrosis (CF) (hereinafter referred to as CF patients) commonly have chronic rhinosinusitis and sinonasal polyposis. Studies have demonstrated histopathologic differences in the paranasal sinus mucosa and sinonasal polyp architecture in CF patients compared with patients who have sinonasal disease but not CF.1 However, the underlying pathophysiological features of polypoid rhinosinusitis remain ill defined in both of these groups.

Dysfunction of the arachidonic acid pathway has been implicated in the pathogenesis of sinonasal polyposis in patients who do not have CF, particularly those with associated asthma and aspirin intolerance.2,3 The prostaglandins (PGs) and leukotrienes (LTs) are the main products of the arachidonic acid pathway, and each appears to have wide-reaching influence on the development and regulation of inflammatory disease in the respiratory system. Prostaglandins are generated through the cyclooxygenase (COX) pathway of arachidonic acid metabolism, and LTs are synthesized through the lipoxygenase (LO) pathway (Figure 1). The COX pathway consists of 2 primary enzymes, ubiquitously expressed COX-1 and inducible COX-2. The LO pathway consists of the 3 primary pathways of 5-LO, 12-LO, and 15-LO. There is accumulating evidence that low PG production combined with upregulated LT synthesis may be important in the development of airway inflammation and subsequent sinonasal polyp formation.2,4 Studies on upper airway respiratory epithelium from patients who do not have CF have suggested that altered regulation of COX-1 and COX-2 may play an important role in the development of airway inflammation and sinonasal polyposis.2,5 The products of the LO pathway are known to be important mediators in airway inflammation; however, our understanding of LO expression remains limited.3,4

Place holder to copy figure label and caption
Figure 1.

Staining for cyclooxygenase 2 (COX-2) in a specimen from a patient with cystic fibrosis. The columnar epithelium displays 3+ staining of apical cytoplasm and 1+ staining of basal cytoplasm. The submucosal glands exhibit 1+ cytoplasmic staining (COX-2 monoclonal antibody, original magnification ×20).

Graphic Jump Location

A considerable volume of literature has been devoted to describing the inflammatory and immunologic mediators in the pathophysiologic mechanisms of airway disease in CF. Most of these efforts have focused on the lower respiratory system,6,7 with very limited published data available exploring these mediators in sinonasal tissue.8,9 With regard to the arachidonic acid pathway, a similar trend is noted in the literature. The presence of PGs and LTs have been explored in the lower respiratory system, urine, and peripheral leukocytes of CF patients. Levels of PGE2, LTB4, and LTE4 have been noted to be elevated in the saliva, sputum, and urine of CF patients relative to those of healthy control subjects.1013 Furthermore, sputum LT levels have been shown to correlate with severity of pulmonary disease in CF patients.14 These data implicate the COX and LO pathways in the pathophysiologic mechanisms of pulmonary disease in CF.

The role of the arachidonic pathway and its metabolites in the development of sinonasal disease in the CF population has not been defined. The objective of this study was to use immunohistochemical techniques to evaluate the expression of the COX (COX-1 and COX-2) and LO (5-LO, 12-LO, and 15-LO) pathway enzymes in sinonasal mucosa from CF patients.

SPECIMEN SELECTION

After receiving approval from the Colorado institutional review board, archival, formalin-fixed tissue blocks of paranasal sinus tissue specimens from CF patients were identified by review of the surgical pathology databases of the University of Colorado Hospital and The Children's Hospital, Denver. Nine CF mucosal biopsy specimens were selected for inclusion in the study, based on confirmation of adequate tissue for analysis by review of the hematoxylin-eosin–stained sections. All 9 patients had nasal polyposis or hyperplastic rhinosinusitis documented by the operating surgeon. All samples were collected from the ethmoid sinus for the CF and control groups. These patients ranged in age from 5 to 51 years. Four patients undergoing endoscopic orbital decompression or nasal surgery for nasal obstruction without rhinosinusitis were also identified as a control group. Preoperative computed tomographic scans of the sinus were graded according to the Lund-McKay rhinosinusitis staging system. Patient records were queried for preoperative oral and topical corticosteroid use and dosage.

IMMUNOHISTOCHEMICAL ANALYSIS

Immunohistochemical reagents included murine COX-1 monoclonal antibody to sheep seminal vesicle COX-1, murine COX-2 monoclonal antibody to a polypeptide sequence from human COX-2, rabbit 5-LO polyclonal antibody to a peptide from human 5-LO, rabbit 12-LO polyclonal antibody to murine 12-LO, and sheep 15-LO polyclonal antibody to rabbit reticulocyte 15-LO (Cayman Chemical Co, Ann Arbor, Michigan). Tissue samples in 5-μm sections were mounted on charged glass slides (Superfrost Plus; Fisher Scientific, Pittsburgh, Pennsylvania) and baked overnight at 60°C. Deparaffinization with xylene and rehydration through a graded alcohol series was followed by the blocking of endogenous peroxidase activity with 3.0% hydrogen peroxide for 15 minutes. Antigen retrieval was performed by heating in a decloaking chamber (Biocare Medical, Walnut Creek, California) in citrate buffer (20 mmol/L [pH, 6.0]) at 120°C for 10 minutes. Staining was performed using an autostainer (Dakocytomation, Carpinteria, California). An indirect avidin-biotin immunoperoxidase method (Vector Laboratories, Burlingame, California) was used following the manufacturer's protocol for all cases except 15-LO, for which a biotinylated anti–sheep IgG (Sigma-Aldrich Corp, St Louis, Missouri) replaced the vector IgG. For COX-1, ovarian carcinoma tissue was used as the control and the final antibody dilution was 5 μg/mL. For COX-2, prostate adenocarcinoma tissue was used, with a final antibody dilution of 1 μg/mL. Colon adenocarcinoma tissue was used as the control for 5-LO and 15-LO, with final antibody dilutions of 1:1500 and 1:250, respectively. The control tissue used for 12-LO was prostate adenocarcinoma, with a final antibody dilution of 1:1000. Paired serial sections were incubated at room temperature for 45 minutes with each particular antibody. Negative control staining was performed on all sections using equivalent concentrations of subclass-matched IgG18 (Becton Dickson Pharmingen, Franklin Lakes, New Jersey) generated against unrelated antigens for COX-1 and COX-2, rabbit serum for 5-LO and 12-LO, and anti–sheep IgG for 15-LO. Enzyme expression was visualized by development with 3,3′-diaminobenzidine (Dakocytomation), counterstained with hematoxylin, dehydrated in graded alcohols, and placed under a coverslip.

Blocking Studies

To ensure the specificity of staining, blocking studies were performed when possible. Blocking peptides for the COX-1, COX-2, and 5-LO antibodies were purchased from Cayman Chemical Co. Using a mole-mole excess of 100 for COX-1, 50 for COX-2, and 150 for 5-LO, the blocking peptide was incubated with the primary antibody for 45 minutes at ambient temperature. This was followed by the standard staining protocol described in the preceding subsection. No peptide or intact enzyme was available for blocking studies with 12-LO and 15-LO. Pancreatic tissue was used to perform positive and negative control studies for 12-LO because islet cells are noted to express the enzyme, whereas exocrine pancreas does not. This was confirmed using the standard concentration and staining protocol. No data regarding the tissue specificity for 15-LO expression were identified by review of the literature.

Grading

Each slide was evaluated by a pathologist (K.R.S.), and the staining intensity for each enzyme was scored on a scale of 0 to 3+ in the columnar epithelium, submucosal glands, and stromal cellular components, based on a review of all histologic sections. In cases with variable levels of staining, scores reflected the peak levels of staining. The median staining intensity scores were determined for each enzyme and were compared between the CF and control groups by means of the Mann-Whitney test, using SPSS statistical software (SPSS Inc, Chicago, Illinois).

The patient demographic data are reported in Table 1. Six male and 3 female CF patients were included in the study, ranging in age from 5 to 51 years. The available data regarding preoperative corticosteroid use were inconsistently documented in the medical records and were thus excluded from evaluation. No patient in either group had documentation of aspirin sensitivity. Lund-McKay staging scores for CF and control patients are reported in Table 1. The scores for the CF patients ranged from 9 to 24 (median, 22). Computed tomographic scans were available for 3 of the control patients, and their Lund-McKay staging scores ranged from 0 to 2 (median, 2). One patient undergoing orbital decompression had a unilateral, completely opacified sphenoid sinus; another patient undergoing septoplasty also had a unilateral, opacified sphenoid sinus. The differences in the scores between groups was statistically significant (P = .01).

Table Graphic Jump LocationTable 1. Patient Demographics and Selected Data

Characteristic and reproducible patterns of staining were observed for each enzyme. The specificity of this staining was confirmed by evaluation of negative control studies and by blocking studies. COX-1 was localized to the cytoplasm of all epithelial and glandular cells. COX-2 was expressed in the plasma membrane and cytoplasm of epithelial and glandular cells, with more intense staining noted in the cytoplasm of more apical epithelial cell layers (Figure 1). Localization of 5-LO to the cytoplasm of epithelial and glandular cells was observed, with more intense staining of the cytoplasm of basal layers of epithelium. Staining for 12-LO was found in the cytoplasm and nucleus of epithelial and glandular cells, with homogeneous full-thickness staining of the epithelium (Figure 2). Staining for 15-LO was observed in the cytoplasm of epithelial and glandular cells, with full-thickness epithelial staining noted. Some nuclear staining was also noted in columnar epithelium. Submucosal glands were not present in 2 CF patients; thus, 7 specimens were included in these comparisons. The statistically significant results are summarized in Table 2. Given the nonparametric nature of the scoring data, representative median values are reported for each group rather than mean values.

Place holder to copy figure label and caption
Figure 2.

Staining for 12-lipoxygenase (12-LO) in a specimen from a patient with cystic fibrosis. The columnar epithelium exhibits 2+ full-thickness cytoplasmic and nuclear staining. The submucosal glands exhibit 2+ cytoplasmic and 3+ nuclear staining (12-LO monoclonal antibody, original magnification ×20).

Graphic Jump Location
Table Graphic Jump LocationTable 2. Staining Scores for COX-1, COX-2, and 12-LO Between the CF and Control Groups
CYCLOOXYGENASE 1

COX-1 expression was detected in columnar epithelium in 3 of 9 CF specimens (33%), with no staining in the control specimens. When present, staining for COX-1 exhibited nearly uniform cytoplasm staining of the full thickness of the epithelium. The median value of the staining was 0 (range, 0-1+). No statistically significant differences in columnar epithelial staining were noted between the groups.

In the submucosal glands, staining for COX-1 was noted in 3 of 7 CF specimens (43%) and 1 of 4 control specimens (25%). Staining was noted in the glandular cytoplasm of these specimens. For each group, the median value of staining was 0 (range, 0-1+). No statistically significant differences were noted between the groups.

CYCLOOXYGENASE 2

Expression of COX-2 in columnar epithelium was noted in all 9 of the CF specimens (100%) and none of the control specimens. In the CF specimens, staining for COX-2 was commonly noted, with a predominance toward staining the apical one-third of the columnar epithelium. When the basal cell layers of epithelium were stained, the intensity of staining was predominantly localized to the apical cell cytoplasm. Among the CF specimens, plasma membrane staining was noted in 5 of 9 specimens (56%); apical cell layer cytoplasm staining was noted in all 9 (100%); and basal cell layer cytoplasm staining was noted in 3 of 9 (33%). The median score for cytoplasm staining was 2+ (range, 2-3+), whereas the median plasma membrane staining score was 1+ (range, 0-3+). No nuclear staining was noted. The difference in the staining of the epithelial cytoplasm was statistically significant (P = .001).

COX-2 was expressed in the submucosal glands of 6 of 7 CF specimens (86%) and 2 of 4 control specimens (50%). Among the CF specimens, plasma membrane staining was present in 2 of 7 specimens (29%) and cytoplasm staining was noted in 6 of 7 (86%), with no nuclear staining noted. Control specimens demonstrated plasma membrane staining in 2 of 4 specimens (50%), with no cytoplasm or nuclear staining. For each group, the median value of plasma membrane staining was 0 (range, 0-2+). Cytoplasm staining in CF specimens had a median score of 2+ (range, 0-2+). The difference in staining between the 2 groups was statistically significant (P = .01).

5-LIPOXYGENASE

Expression of 5-LO was noted in all specimens in each group. Expression was confined to the cytoplasm in columnar epithelium and submucosal glands because no plasma membrane or nuclear staining was seen. Epithelial staining was more intense in the basal cell layers of the epithelium than in the apical cell layers. In columnar epithelium, CF specimens demonstrated staining of the apical cell layer cytoplasm and basal cytoplasm in all specimens. Control specimens demonstrated apical cell layer cytoplasm staining in 2 of 4 specimens (50%) and basal cytoplasm staining in all 4 (100%). For each group, the cytoplasm staining median value was 2+, with CF specimens having a range of 0 to 3+ and control specimens, of 2 to 3+. No statistically significant differences in the staining of epithelium were noted between the groups.

Cytoplasm staining for 5-LO in submucosal glands was present in all CF specimens and in 3 of 4 control specimens (75%). The median values were 2+ for CF specimens (range, 1-2+) and 1+ for control specimens (range, 0-2+). No statistically significant differences were noted between the groups.

12-LIPOXYGENASE

In columnar epithelium, 12-LO was present in all of the CF specimens and in 3 of 4 control specimens (75%). No plasma membrane staining was noted. The CF specimens demonstrated apical and basal cell layer cytoplasm staining in all specimens and nuclear staining in 8 of 9 (89%). The median score for cytoplasm staining of the CF specimens was 2+ (range, 2-3+), whereas the median nuclear staining score was 2+ (range, 0-3+). Control specimens exhibited apical cytoplasm staining in 2 of 4 specimens (50%), basal cell layer cytoplasm staining in 3 of 4 (75%), and nuclear staining in none. The median value for cytoplasm staining in controls was 1+ (range, 0-2+). Statistically significant differences were noted in the staining of the apical cell layer cytoplasm (P = .03), basal cell layer cytoplasm (P = .03), and nucleus (P = .009).

Staining for 12-LO was noted in submucosal glands in all of the CF specimens and in 1 of 4 control specimens (25%). No plasma membrane staining was noted. Staining for 12-LO was observed in the cytoplasm of all of the CF specimens and in the nucleus of 4 of 7 specimens (57%), with a median value of 2+ (range, 1-3+). Control specimens demonstrated cytoplasm staining in 1 of 4 specimens (25%) and nuclear staining in 1 of 4 (25%). The median score of cytoplasm staining was 0 (range, 0-2+); the median score for nuclear staining was 0 (range, 0-3+). A statistically significant difference in cytoplasm staining (P = .045) was noted between the groups.

15-LIPOXYGENASE

Columnar epithelium staining for 15-LO was exhibited by all specimens in each group. No plasma membrane staining was noted. Among the CF specimens, apical and basal cell layer cytoplasm staining was noted in all specimens, whereas no nuclear staining was noted. The median value of cytoplasm staining was 2+ (range, 1-3+). Control specimens demonstrated apical cell cytoplasm staining in 2 of 4 specimens (50%), basal cell cytoplasm staining in all specimens, and nuclear staining in 1 of 4 (25%). Median cytoplasm staining was 2+ (range, 1-3+). No statistically significant differences were noted between the groups.

Submucosal gland staining for 15-LO was noted in 4 of 7 CF specimens (57%) and in 1 of 4 control specimens (25%). Staining was confined to the cytoplasm in all specimens expressing the enzyme. Median values were 1+ (range, 0-2+) for CF specimens and 0 (range, 0-2+) for control specimens. No statistically significant differences were noted between the groups.

This study defined the expression of COX and LO enzymes in sinonasal tissue from CF patients. Although studies have demonstrated altered LT and PG levels in the urine, sputum, and epithelial lining fluid of CF patients, the roles of the COX and LO enzymes in the upper airway have not been as well defined. Specifically, COX-1 and COX-2 expression in sinonasal tissue from CF patients has only recently been investigated in a single publication.9 Our data are consistent with recent studies that have indicated that dysregulation of COX-1 and COX-2, and possibly the LO enzymes, may be important in the pathogenesis of polypoid rhinosinusitis in patients with and without CF.2,3

The present study did not identify any difference in the expression of COX-1 in the CF group compared with controls. Cyclooxygenase-1 represents the constitutive isoform and performs a housekeeping function to synthesize PGs that regulate normal cell activity, and it is generally accepted that its expression does not vary greatly.2,15 Thus, our findings are consistent with those of previous studies and the conceptual understanding of COX-1 in inflammatory disease in patients who do not have CF.16 Kuitert et al17 evaluated the expression of several arachidonic acid pathway mediators in mononuclear and polymorphonuclear cells in blood from CF patients using a semiquantitative technique. They did not find differences in the expression of COX-1 in the CF compared with control tissue. Although they investigated peripheral blood cells, their results parallel our findings in sinonasal tissue and support the hypothesis that COX-1 is a constitutive enzyme. More recently, Roca-Ferrer et al9 reported increased COX-1 expression in polyp tissue from CF patients compared with tissue from healthy controls.

Cyclooxygenase 2 is the inducible isoform of the enzyme, and its expression can increase in response to a variety of stimuli, including cytokines and growth factors.18 Cyclooxygenase 2 is partly responsible for the production of the PGs, which are believed to have proinflammatory and anti-inflammatory functions. We found statistically significant differences in COX-2 expression between CF and control sinonasal mucosa in our study. Increased expression of COX-2 was demonstrated in the columnar epithelium and the submucosal glands in the CF group, with a predominance toward staining in the apical cytoplasm without evidence of nuclear staining. The significance of this polarity of epithelial staining is uncertain. Only 1 previously reported study has investigated COX-2 expression in polyp tissue from CF patients. In that study, Roca-Ferrer et al9 also reported an increase in COX-2 expression in polyp tissue of CF patients compared with that of normal controls. Our findings are consistent with that group's data. Kuitert et al17 failed to detect any baseline expression of COX-2 in peripheral blood cells from CF patients. A growing body of work has investigated COX-2 expression in sinonasal tissue of patients who do not have CF. Using similar immunohistochemical techniques, the data from these studies have produced inconsistent results. Demoly et al19 reported no difference in the immunoreactivity of COX-2 in sinonasal epithelium of healthy controls compared with patients with rhinitis, sinonasal polyps, and chronic sinusitis. Additional work failed to demonstrate differences in COX-2 expression in bronchial tissue from healthy controls vs patients with asthma.20 However, other groups have reported increased immunoreactivity of COX-2 in patients with asthma compared with healthy controls, findings similar to ours.16,21,22 The expression of COX-2 appears to vary between patient groups, thus suggesting a different role in the CF patient compared with the patient with polyps who does not have CF.

The experimental methods may be important when trying to understand these conflicting data. Recent work was performed by Pujols et al2 in which dynamic molecular techniques were used to demonstrate differential kinetics of COX-2 messenger RNA between normal sinonasal mucosa and sinonasal polyp tissue in aspirin-tolerant and aspirin-intolerant patients. They demonstrated a downregulation of COX-2, particularly in aspirin-intolerant patients. This finding supports the hypothesis that COX-2 dysregulation leading to decreased PGE2 production may be a critical factor in the development of airway inflammation in some patient groups. Immunohistochemical techniques are limited in that only a single time is evaluated, whereas dynamic methods assess the adaptive response of the enzyme. Dynamic methods could not be used in this study because we studied archived tissue. With the current lack of literature exploring the role of COX-2 in CF, it is difficult, if not impossible, to speculate on the significance of our findings and how they might correlate with the recent work on respiratory tissue from patients who do not have CF. Based on our data, it is plausible to speculate that the dysregulation of COX-2 may be important in the pathogenesis of sinonasal disease in CF patients; however, it remains unclear whether this is owing to variable enzyme expression and function or a consequence of posttranscriptional modifications to the enzymes. Further work in this area is required to understand these potential differences.

The LO pathway is the other arm of the arachidonic acid metabolic pathway and is responsible for the production of the LTs. The enzyme 5-LO, together with 5-LO activating protein (FLAP), generates LTA4 from arachidonic acid. Then, through the action of LTC4 synthase, the proinflammatory products LTC4, LTD4, and LTE4 are synthesized. Similar to the published work on the COX pathways, the current literature investigating the LO pathways has been restricted primarily to the lower airway, with limited data based on sinonasal tissue. In addition, we were unable to identify previous studies exploring the role of the LO enzymes in CF sinonasal tissue.

Elevated levels of LTs have been found in the urine, sputum, serum, and bronchial lavage fluid of CF patients.1013,23 To the best of our knowledge, neither sinonasal tissue levels of LTs nor LO expression has been investigated in this population. We did not find a difference in 5-LO expression between the CF and control groups. These findings are similar to the study by Kuitert et al,17 which demonstrated equivalent levels of 5-LO expression in peripheral blood cells from CF patients compared with controls. These data suggest that elevated 5-LO expression is not primarily responsible for increased LT production and may highlight the importance of posttranscriptional effects. It has been shown that the interaction between 5-LO and FLAP, a nuclear membrane protein, is a critical rate-limiting step in the activation of LT production by 5-LO.24 Therefore the posttranscriptional effects of regulatory factors such as FLAP may be more important than the absolute expression of 5-LO in the abnormal production of LTs. Our data finding equal expression of 5-LO in CF sinonasal tissue compared with that of controls are consistent with this concept. Future studies specifically addressing possible posttranscriptional regulatory factors will increase our understanding of this process.

The biological role of 15-LO and its products remains poorly defined. It has been established that 15-LO is important in cell differentiation and maturation, inflammation, asthma, carcinogenesis, and atherogenesis. The products of 15-LO metabolism were initially believed to be proinflammatory; however, in recent years several lines of experimental evidence have suggested that anti-inflammatory properties might be more important.25 The main product of 15-LO metabolism is 15-hydroxyeicosatetraenoic acid (15-HETE), which has been associated with bronchial mucus production, bronchial smooth muscle contraction, and vascular permeability. The precise role of 15-LO in airway inflammation, however, remains unclear. Several studies have confirmed elevated expression of 15-LO in human lower airways, specifically in patients with asthma and atopy.25 We did not find any difference in the expression of 15-LO in our CF group compared with normal controls. The study by Kuitert et al17 is the only published work we identified that has looked at 15-LO expression in CF patients. They examined peripheral blood cells and failed to detect any expression of 15-LO in the CF group, but did measure an increased level of expression in cells from patients with asthma compared with controls. The absence of elevated expression of 15-LO in our results and the data from Kuitert et al are interesting. Together these results suggest that both peripheral inflammatory cells and activated cells in sinonasal tissue may fail to show 15-LO activity in response to inflammation. This finding may suggest a dysregulation in the anti-inflammatory response leading to or associated with altered 15-LO activity and ultimately contributing to airway inflammation.

The role of 12-LO and its major product, 12-HETE, is even less well defined in airway inflammation. A thorough search of MEDLINE identified a single report examining 12-LO expression in human sinonasal epithelium.3 We found increased expression of 12-LO in the CF specimens compared with controls in both the columnar epithelium and submucosal glands. The staining of epithelium for 12-LO was full thickness and lacked the polarity noted for COX-2 and 5-LO. The significance of this finding is uncertain. To the best of our knowledge, this is the first report of 12-LO expression in CF sinonasal epithelium. The biochemical and biological properties of 12-LO and 15-LO appear to be similar but their tissue specificity and location differ. Expression of 12-LO has been well described in bovine airway epithelium, whereas 15-LO has been more extensively studied in human airway epithelium.26 The involvement of 12-LO expression in carcinogenesis, particularly metastasis, cellular apoptosis, and oxidative stress, has been reported previously.2730 Studies have also shown that the helper T-cell subtype 2–derived cytokines upregulate murine macrophage 12-LO expression in vivo and in vitro.31 The production of the primary product of 12-LO, 12-HETE, has been demonstrated to be inducible from platelets and granulocytes of CF patients after exposure to Pseudomonas products phospholipase C, lipase, and glycolipid.32 Despite intensive research on the expression and function of 12-LO in tissue systems other than respiratory epithelium, our understanding of the biological significance of this enzyme in the airway inflammation is limited.27

Several limitations of this study must be discussed. Because we were working with archived tissue, we were not able to use real-time polymerase chain reaction techniques as described by Pujols et al.2 Immunohistochemical staining techniques provide description of enzyme expression at a single time, not allowing for evaluation of that enzyme's ability to adapt. With select enzymes that appear to be inducible isoforms, such as COX-2, this difference in methods may be important. Immunohistochemical techniques are important in defining the presence and location of the enzymes, and dynamic techniques help to further elucidate the biological properties of these select targets. Thus, these techniques are complimentary. An additional limitation of this study is a consequence of the retrospective method in which the tissue and patients were identified. Corticosteroids are known to suppress the arachidonic metabolic pathway to varying degrees in different patient groups. We were not able to control for oral or nasal corticosteroid use in our study patients; thus, the potential impact of these medications on our results is not clear. Our sample size is obviously small and may not be powered sufficiently. At the very least, this study serves as a starting point to describe the expression of enzymes not previously explored in CF sinonasal tissue. Finally, it was not our intent to characterize the precise inflammatory cell populations responsible for enzyme expression in the study tissue.

In conclusion, immunohistochemical techniques were used in this study to define the expression of the COX and LO pathway enzymes in sinonasal tissue from CF patients. We found significant differences in the expression of COX-2 and 12-LO in CF tissue compared with control tissue. The expression of 5-LO, COX-1, and 15-LO did not appear to differ between the 2 groups. Our data suggest that dysregulation of the arachidonic pathway may be important in the pathogenesis of upper airway inflammation in the CF patient and warrants further investigation.

Correspondence: Todd T. Kingdom, MD, Department of Otolaryngology–Head and Neck Surgery, University of Colorado, 12631 E 17th Ave, B-205, PO Box 6511, Aurora, CO 80045 (todd.kingdom@uchsc.edu).

Submitted for Publication: May 3, 2007; final revision received October 8, 2007; accepted October 15, 2007.

Author Contributions: Drs Owens and Kingdom had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Study concept and design: Owens, Shroyer, and Kingdom. Acquisition of data: Shroyer and Kingdom. Analysis and interpretation of data: Owens, Shroyer, and Kingdom. Drafting of the manuscript: Owens and Kingdom. Critical revision of the manuscript for important intellectual content: Owens, Shroyer, and Kingdom. Statistical analysis: Owens. Administrative, technical, and material support: Shroyer and Kingdom. Study supervision: Shroyer and Kingdom.

Financial Disclosure: None reported.

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Smith  WLDewitt  DL Prostaglandin endoperoxide H synthases-1 and -2. Adv Immunol 1996;62167- 215
PubMed
Sousa  APfister  RChristie  PE  et al.  Enhanced expression of cyclo-oxygenase isoenzyme 2 (COX-2) in asthmatic airways and its cellular distribution in aspirin-sensitive asthma. Thorax 1997;52 (11) 940- 945
PubMed Link to Article
Kuitert  LMNewton  RBarnes  NCAdcock  IMBarnes  PJ Eicosanoid mediator expression in mononuclear and polymorphonuclear cells in normal subjects and patients with atopic asthma and cystic fibrosis. Thorax 1996;51 (12) 1223- 1228
PubMed Link to Article
Fernández-Morata  JCMullol  JFuentes  M  et al.  Regulation of cyclooxygenase-1 and -2 expression in human nasal mucosa: effects of cytokines and dexamethasone. Clin Exp Allergy 2000;30 (9) 1275- 1284
PubMed Link to Article
Demoly  PCrampette  LLebel  BCampbell  AMMondain  MBousquet  J Expression of cyclo-oxygenase 1 and 2 proteins in upper respiratory mucosa. Clin Exp Allergy 1998;28 (3) 278- 283
PubMed Link to Article
Demoly  PJaffuel  DLequeux  N  et al.  Prostaglandin H synthase 1 and 2 immunoreactivities in the bronchial mucosa of asthmatics. Am J Respir Crit Care Med 1997;155 (2) 670- 675
PubMed Link to Article
Redington  AEMeng  QHSpringall  DR  et al.  Increased expression of inducible nitric oxide synthase and cyclo-oxygenase-2 in the airway epithelium of asthmatic subjects and regulation by corticosteroid treatment. Thorax 2001;56 (5) 351- 357
PubMed Link to Article
Taha  ROlivenstein  RUtsumi  T  et al.  Prostaglandin H synthase 2 expression in airway cells from patients with asthma and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2000;161 (2, pt 1) 636- 640
PubMed Link to Article
Lee  THCrea  AEGant  V  et al.  Identification of lipoxin A4 and its relationship to the sulfidopeptide leukotrienes C4, D4, and E4 in the bronchoalveolar lavage fluids obtained from patients with selected pulmonary disease. Am Rev Respir Dis 1990;141 (6) 1453- 1458
PubMed Link to Article
Peters-Golden  M Cell biology of the 5-lipoxygenase pathway. Am J Respir Crit Care Med 1998;157 (6) S227- S232
PubMed Link to Article
Kuhn  HWalther  MKuban  RJ Mammalian arachidonate 15-lipoxygenases: structure, function, and biologic activity. Prostaglandins Other Lipid Mediat August2002;68-69263- 290
PubMed Link to Article
De Marzo  NSloane  DLDicharry  SHighland  ESigal  E Cloning and expression of an airway epithelial 12-liopxygenase. Am J Physiol 1992;262 (2, pt 1) L198- L207
PubMed
Yoshimoto  TTakahashi  Y Arachidonate 12-lipoxygenases. Prostaglandins Other Lipid Mediat 2002;68-69245- 262
PubMed Link to Article
Yoshimura  RInoue  KKawahito  Y  et al.  Expression of 12-lipoxygenase in human renal cell carcinoma and growth prevention by its inhibitor. Int J Mol Med 2004;13 (1) 41- 46
PubMed
Wang  HLi  JFollett  PL  et al.  12-Lipoxygenase plays a key role in cell death caused by glutathione depletion and arachidonic acid in rat oligodendrocytes. Eur J Neurosci 2004;20 (8) 2049- 2058
PubMed Link to Article
Gillis  RCDaley  BJEnderson  BLKarlstad  MD Role of downstream metabolic processing of proinflammatory fatty acids by 5-lipoxygenase in HL-60 cell apoptosis. J Trauma 2003;54 (1) 91- 103
PubMed Link to Article
Heydeck  DThomas  LSchnurr  K  et al.  Interleukin-4 and –13 induce upregulation of the murine macrophage 12/15-lipoxygenase activity: evidence for the involvement of transcription factor STAT6. Blood 1998;92 (7) 2503- 2510
PubMed
König  BJaeger  KEKonig  W Induction of inflammatory mediator release (12-hydroxyeicosatetraenoic acid) from human platelets by Pseudomonas aeruginosaInt Arch Allergy Immunol 1994;104 (1) 33- 41
PubMed Link to Article

Figures

Place holder to copy figure label and caption
Figure 1.

Staining for cyclooxygenase 2 (COX-2) in a specimen from a patient with cystic fibrosis. The columnar epithelium displays 3+ staining of apical cytoplasm and 1+ staining of basal cytoplasm. The submucosal glands exhibit 1+ cytoplasmic staining (COX-2 monoclonal antibody, original magnification ×20).

Graphic Jump Location
Place holder to copy figure label and caption
Figure 2.

Staining for 12-lipoxygenase (12-LO) in a specimen from a patient with cystic fibrosis. The columnar epithelium exhibits 2+ full-thickness cytoplasmic and nuclear staining. The submucosal glands exhibit 2+ cytoplasmic and 3+ nuclear staining (12-LO monoclonal antibody, original magnification ×20).

Graphic Jump Location

Tables

Table Graphic Jump LocationTable 1. Patient Demographics and Selected Data
Table Graphic Jump LocationTable 2. Staining Scores for COX-1, COX-2, and 12-LO Between the CF and Control Groups

References

Oppenheimer  EHRosenstein  BJ Differential pathology of nasal polyps in cystic fibrosis and atopy. Lab Invest 1979;40 (4) 445- 449
PubMed
Pujols  LMullol  JAlobid  IRoca-Ferrer  JXaubet  APicado  C Dynamics of COX-2 in nasal mucosa and nasal polyps from aspirin-tolerant and aspirin-intolerant patients with asthma. J Allergy Clin Immunol 2004;114 (4) 814- 819
PubMed Link to Article
Owens  JMShroyer  KRKingdom  TT Expression of cyclooxygenase and lipoxygenase enzymes in nasal polyps of aspirin-sensitive and aspirin-tolerant patients. Arch Otolaryngol Head Neck Surg 2006;132 (6) 579- 587
PubMed Link to Article
Babu  KSSalvi  SS Aspirin and asthma. Chest 2000;118 (5) 1470- 1476
PubMed Link to Article
Mullol  JFernandez-Morata  JCRoca-Ferrer  J  et al.  Cyclooxygenase 1 and cyclooxygenase 2 expression is abnormally regulated in human nasal polyps. J Allergy Clin Immunol 2002;109 (5) 824- 830
PubMed Link to Article
Bonfield  TLKonstan  MWBerger  M Altered respiratory epithelial cell cytokine production in cystic fibrosis. J Allergy Clin Immunol 1999;104 (1) 72- 78
PubMed Link to Article
Bonfield  TLPanuska  JRKonstan  MW  et al.  Inflammatory cytokines in cystic fibrosis lungs [published correction appears in Am J Respir Crit Care Med. 1996;154(4, pt 1):following 1217]. Am J Respir Crit Care Med 1995;152 (6, pt 1) 2111- 2118
PubMed Link to Article
Sobol  SEChristodoulopoulos  PManoukian  JJ  et al.  Cytokine profile of chronic sinusitis in patients with cystic fibrosis. Arch Otolaryngol Head Neck Surg 2002;128 (11) 1295- 1298
PubMed Link to Article
Roca-Ferrer  JPujols  LGartner  S  et al.  Upregulation of COX-1 and COX-2 in nasal polyps in cystic fibrosis. Thorax 2006;61 (7) 592- 596
PubMed Link to Article
Rigas  BKorenberg  JRMerrill  WWLevine  L Prostaglandins E2 and E2 alpha are elevated in saliva of cystic fibrosis patients. Am J Gastroenterol 1989;84 (11) 1408- 1412
PubMed
Konstan  MWWalenga  RWHilliard  KAHilliard  JB Leukotriene B4 markedly elevated in the epithelial lining fluid of patients with cystic fibrosis. Am Rev Respir Dis 1993;148 (4, pt 1) 896- 901
PubMed Link to Article
Sampson  APSpencer  DAGreen  CPPiper  PJPrice  JF Leukotrienes in the sputum and urine of cystic fibrosis children. Br J Clin Pharmacol 1990;30 (6) 861- 869
PubMed Link to Article
Zakrzewski  JTBarnes  NCPiper  PJCostello  JF Detection of sputum eicosanoids in cystic fibrosis and in normal saliva by bioassay and radioimmunoassay. Br J Clin Pharmacol 1987;23 (1) 19- 27
PubMed Link to Article
Spencer  DASampson  APGreen  CPCostello  JF Sputum cysteinyl-leukotriene levels correlate with the severity of pulmonary disease in children with cystic fibrosis. Pediatr Pulmonol 1992;12 (2) 90- 94
PubMed Link to Article
Smith  WLDewitt  DL Prostaglandin endoperoxide H synthases-1 and -2. Adv Immunol 1996;62167- 215
PubMed
Sousa  APfister  RChristie  PE  et al.  Enhanced expression of cyclo-oxygenase isoenzyme 2 (COX-2) in asthmatic airways and its cellular distribution in aspirin-sensitive asthma. Thorax 1997;52 (11) 940- 945
PubMed Link to Article
Kuitert  LMNewton  RBarnes  NCAdcock  IMBarnes  PJ Eicosanoid mediator expression in mononuclear and polymorphonuclear cells in normal subjects and patients with atopic asthma and cystic fibrosis. Thorax 1996;51 (12) 1223- 1228
PubMed Link to Article
Fernández-Morata  JCMullol  JFuentes  M  et al.  Regulation of cyclooxygenase-1 and -2 expression in human nasal mucosa: effects of cytokines and dexamethasone. Clin Exp Allergy 2000;30 (9) 1275- 1284
PubMed Link to Article
Demoly  PCrampette  LLebel  BCampbell  AMMondain  MBousquet  J Expression of cyclo-oxygenase 1 and 2 proteins in upper respiratory mucosa. Clin Exp Allergy 1998;28 (3) 278- 283
PubMed Link to Article
Demoly  PJaffuel  DLequeux  N  et al.  Prostaglandin H synthase 1 and 2 immunoreactivities in the bronchial mucosa of asthmatics. Am J Respir Crit Care Med 1997;155 (2) 670- 675
PubMed Link to Article
Redington  AEMeng  QHSpringall  DR  et al.  Increased expression of inducible nitric oxide synthase and cyclo-oxygenase-2 in the airway epithelium of asthmatic subjects and regulation by corticosteroid treatment. Thorax 2001;56 (5) 351- 357
PubMed Link to Article
Taha  ROlivenstein  RUtsumi  T  et al.  Prostaglandin H synthase 2 expression in airway cells from patients with asthma and chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2000;161 (2, pt 1) 636- 640
PubMed Link to Article
Lee  THCrea  AEGant  V  et al.  Identification of lipoxin A4 and its relationship to the sulfidopeptide leukotrienes C4, D4, and E4 in the bronchoalveolar lavage fluids obtained from patients with selected pulmonary disease. Am Rev Respir Dis 1990;141 (6) 1453- 1458
PubMed Link to Article
Peters-Golden  M Cell biology of the 5-lipoxygenase pathway. Am J Respir Crit Care Med 1998;157 (6) S227- S232
PubMed Link to Article
Kuhn  HWalther  MKuban  RJ Mammalian arachidonate 15-lipoxygenases: structure, function, and biologic activity. Prostaglandins Other Lipid Mediat August2002;68-69263- 290
PubMed Link to Article
De Marzo  NSloane  DLDicharry  SHighland  ESigal  E Cloning and expression of an airway epithelial 12-liopxygenase. Am J Physiol 1992;262 (2, pt 1) L198- L207
PubMed
Yoshimoto  TTakahashi  Y Arachidonate 12-lipoxygenases. Prostaglandins Other Lipid Mediat 2002;68-69245- 262
PubMed Link to Article
Yoshimura  RInoue  KKawahito  Y  et al.  Expression of 12-lipoxygenase in human renal cell carcinoma and growth prevention by its inhibitor. Int J Mol Med 2004;13 (1) 41- 46
PubMed
Wang  HLi  JFollett  PL  et al.  12-Lipoxygenase plays a key role in cell death caused by glutathione depletion and arachidonic acid in rat oligodendrocytes. Eur J Neurosci 2004;20 (8) 2049- 2058
PubMed Link to Article
Gillis  RCDaley  BJEnderson  BLKarlstad  MD Role of downstream metabolic processing of proinflammatory fatty acids by 5-lipoxygenase in HL-60 cell apoptosis. J Trauma 2003;54 (1) 91- 103
PubMed Link to Article
Heydeck  DThomas  LSchnurr  K  et al.  Interleukin-4 and –13 induce upregulation of the murine macrophage 12/15-lipoxygenase activity: evidence for the involvement of transcription factor STAT6. Blood 1998;92 (7) 2503- 2510
PubMed
König  BJaeger  KEKonig  W Induction of inflammatory mediator release (12-hydroxyeicosatetraenoic acid) from human platelets by Pseudomonas aeruginosaInt Arch Allergy Immunol 1994;104 (1) 33- 41
PubMed Link to Article

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