554944
research-article2014
AORXXX10.1177/0003489414554944Annals of Otology, Rhinology & LaryngologyPerić et al
Article
Macrophage Inflammatory Protein-1 Production and Eosinophil Infiltration in Chronic Rhinosinusitis With Nasal Polyps
Annals of Otology, Rhinology & Laryngology 2015, Vol. 124(4) 266–272 © The Author(s) 2014 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/0003489414554944 aor.sagepub.com
Aleksandar Perić, MD, PhD1, Nenad Baletić, MD, PhD1, Jelena Sotirović, MD1, and Cveta Špadijer-Mirković, MD2
Abstract Objective: Eosinophil recruitment to the nasal mucosa involves a number of chemokines. The aim of this study was to evaluate nasal secretion levels of macrophage inflammatory protein-1 alpha (MIP-1α) and MIP-1β and to correlate these levels with clinical characteristics and degree of eosinophilia in nonallergic and allergic patients with nasal polyposis (NP). Methods: Fourteen nonatopic and 14 atopic patients with NP were recruited for this cross-sectional study. Fourteen healthy subjects were included as controls. The concentrations of MIP-1α and MIP-1β in nasal secretions were measured by flow cytometry. Eosinophil counts were performed by cytological examination of the scraped nasal mucosa. We scored each of the 28 patients according to the nasal symptom score, endoscopic score, and computed tomography (CT) score. Results: We found significantly higher concentrations of MIP-1α in nasal fluid of nonallergic and allergic NP patients compared to control subjects. In nonallergic patients, we found positive correlations between MIP-1α levels and endoscopic score, CT score, and the percentage of eosinophils. Conclusion: MIP-1α may play a role in eosinophil recruitment in NP. Our results suggest that the measurement of MIP-1α in nasal secretions could be useful in evaluating the degree of eosinophil inflammation and severity of disease in nonallergic patients. Keywords chronic rhinosinusitis, nasal polyps, nasal secretions, eosinophils, chemokines, MIP-1
Introduction Chronic rhinosinusitis (CRS) is a multifactorial inflammatory disease of the nasal and paranasal sinus mucosa, and it can be divided into polypous and nonpolypous forms. Clinical and experimental studies1,2 indicate that nasal polyp growth is conducted by interactions between respiratory epithelium, lamina propria, and inflammatory cells. It may be initiated by both infectious and noninfectious inflammation. The main histological characteristic of chronic rhinosinusitis with nasal polyps (CRSwNP) is subepithelial and epithelial infiltration by eosinophils.3 Several members of the CC chemokine (C-C ligand [CCL]) subfamily, including CCL5 (regulation on activation normal T cell expressed and secreted [RANTES]), CCL7 (monocyte chemoattractant protein-3 [MCP-3]), CCL11 (eotaxin-1), CCL24 (eotaxin-2), and others, play important roles in eosinophil chemotaxis.4 One of these CC chemoattractants, macrophage inflammatory protein-1 (MIP-1), was isolated in 1988 from the supernatant of bacterial endotoxinstimulated murine macrophages. Further biochemical separation and characterization of the protein doublet yielded 2
distinct but highly related proteins, MIP-1α and MIP-1β, that shared 68% identical amino acids.5 Primary sources of these proteoglycan-binding proteins are monocytes, macrophages, and other antigen-presenting cells. After the immunological activation of other immunocompetent cells, MIP-1α and MIP-1β were found to be produced mainly by activated eosinophils and fibroblasts.5 This further MIP-1α/ MIP-1β production could be very important for further eosinophil accumulation in the nasal mucosa and prolongation of chronic inflammation. The concentrations of RANTES, eotaxin-1, and eotaxin-2 in body fluids were associated with the clinical severity of CRS.6,7 However, 1
Department of Otorhinolaryngology, Faculty of Medicine, Military Medical Academy, Belgrade, Serbia 2 Department of Otorhinolaryngology, Health Centre Kosovska Mitrovica, Serbia Corresponding Author: Aleksandar Perić, MD, PhD, Department of Otorhinolaryngology, Rhinology Unit, Faculty of Medicine, Military Medical Academy, Crnotravska 17, 11040 Belgrade, Serbia. Email: [email protected]
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Perić et al the exact role of MIP-1 in sinonasal inflammation has not been fully explored. Previous biochemical and cytological investigations showed that contents of nasal discharge reflect the inflammatory status of the nasal mucosa and the evolution of mucosal diseases.8 The relationship between CRS and allergic rhinitis is controversial. Allergic mucosal edema causes ostial obstruction that interferes with drainage and ventilation from the sinuses, promoting mucus accumulation, serum transduction, decreased oxygenation within the sinuses, and therefore, impaired ciliary movement and bacterial growth.9 The findings presented by Kirtsreesakul and Ruttanaphol9 could suggest an association between allergic rhinitis and rhinosinusitis via IgE mediated hypersensitivity.9 On the other hand, Görgülü et al10 showed no direct relationship between allergy and CRSwNP. Therefore, another study demonstrated clear differences between nonatopic and atopic nasal polyp patients regarding the inflammatory mediator profile in nasal secretions.11 The aim of this study was to evaluate nasal secretion levels of MIP-1α and MIP-1β and to investigate whether or not these concentrations correlate with severity of disease and degree of tissue eosinophilia in CRSwNP patients. The reason for this investigation was also to assess if atopic status of CRS patients has influence on MIP-1α/MIP-1β production and on their chemoattractant activities.
Methods Participants Twenty-eight (n = 28) patients with a diagnosis of CRSwNP were selected for participation in this study. Fourteen (n = 14) patients (8 men and 6 women, mean [SD] age = 40.57 [10.28] years) were nonatopic. The other 14 patients (8 men and 6 women, mean [SD] age = 41.43 [11.18] years) were atopic. This cross-sectional study was performed in accordance with the Declaration of Helsinki. The protocol and methods of the study were approved by the ethics committee of our institution. A written informed consent was obtained from all patients. This study was performed in the rhinology and allergy unit of the department of otorhinolaryngology, Military Medical Academy, Belgrade, Serbia, between September 2012 and December 2013. The diagnosis of CRSwNP was based on guidelines established by the Rhinosinusitis Task Force and endorsed and updated in 2007 by the American Academy of Otolaryngology–Head and Neck Surgery.12,13 The presence of bilateral polyps in the nasal cavities on endoscopic examination and the presence of bilateral areas of opacification in the ethmoidal labyrinths on computed tomography (CT) were the criteria for inclusion in this investigation. Exclusion criteria were the presence of choanal polyps, bronchial asthma, aspirin sensitivity, and systemic diseases affecting the nose (sarcoidosis, primary
ciliary dyskinesia, Wegener’s granulomatosis, cystic fibrosis, and Churg-Strauss syndrome). Also, patients with a history of cigarette smoking and previous paranasal sinus surgery were excluded. None of the patients had any acute upper and lower respiratory tract infections or use of antibiotics, antihistamines, or systemic or topical corticosteroids within 3 weeks before the beginning of this investigation. Fourteen healthy participants, without symptoms, medical history, and endoscopic findings of nasal/paranasal sinus inflammation, were included as controls. The mean [SD] age in the control group (9 male and 5 female participants) was 41.36 [10.05] years.
Allergy Determination The atopic status was evaluated at the beginning of the study by an allergist on the basis of clinical criteria, medical history of allergic rhinitis, positive skin-prick tests, and positive serologic test. Skin-prick tests were performed on the volar part of the forearm with a standard battery of common aeroallergens: birch, timothy, mugwort (Artemisia vulgaris), dog, cat, horse, mite (Dermatophagoides farinae, Dermatophagoides pteronyssinus), and molds (Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Olea europaea, Parietaria judaica, Plantago lanceolata, Platanus acerifolia). Negative (0.9% natrium-chloridum solution) and positive (1 mg/mL histamine dihydrochloride solution) control participants were also included with each skin-prick test. Reactions were read after 15 minutes, and the test was considered positive if the diameter of wheal was greater than 3 mm with respect to the negative control. Total serum IgE level was measured by an ELISA kit (Elitech Diagnostics, Salon-de-Provence, France) and an ELISA reader (Spectra III, Linz, Austria). Venous blood was collected and centrifuged, and the serum stored at −70°C until testing. Participants were considered allergic if they had a serum IgE level > 100 IU/mL.
Clinical Evaluation The same rhinologist examined all patients. All CRSwNP patients were asked to assess their symptoms (nasal obstruction, rhinorrhoea, hyposmia, sneezing, and itching). The symptoms were scored from 0 to 3 (0 for no symptoms, 1 for mild symptoms, 2 for moderate symptoms, and 3 for severe symptoms), resulting in a maximum nasal symptom score of 15, as previously described.14 Endoscopic examination was performed in all CRS patients in a sitting position with a rigid endoscope (0 and 30 degrees; Storz, Tuttlingen, Germany). The nasal polyp size scores were made based on the endoscopic findings, according to Lildholdt et al15: 0 = no polyposis; 1 = mild polyposis (small polyps that do not reach the upper edge of the inferior turbinate); 2 = moderate polyposis (medium-size polyps
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between the upper and lower edges of the inferior turbinate); 3 = severe polyposis (large polyps that reach the lower edge of the inferior turbinate). The maximum endoscopic score is 6, bilaterally. The findings from CT scans were evaluated according to the Lund-Mackay score.16 The mucosal abnormalities were graded as 0 (no abnormality), 1 (partial opacification), or 2 (total opacification) of the frontal, maxillary, anterior ethmoid, posterior ethmoid, and sphenoid sinus, bilaterally. The ostiomeatal complexes were scored bilaterally as 0 (not occluded) or 2 (occluded). The maximal CT grading score is 24.
Sampling of Nasal Secretions and Chemokine Measurement Nasal secretion samples were collected from nasal cavities of all 42 participants—28 CRS patients (14 nonallergic and 14 allergic) and 14 healthy participants—using a modified absorption technique with cotton-wool sticks (length 10 mm, diameter 4 mm; Institute of Virology, Vaccines and Sera, Torlak, Belgrade, Serbia), which were inserted for 5 minutes by bayonette forceps into the anterior part of the middle meatus, under endoscopic guidance, as previously described.11,17 All samples were placed in a 2-mL Eppendorf tube containing 1 mL of transfer medium (phosphate-buffered saline with gentamicin 50 μg/mL, penicillin G 340 IU/mL, fungizone 500 μg/mL) for 30 minutes to allow the diffusion of chemokines into the medium and then stored at 4°C for a maximum of 2 hours until processed. Nasal fluid was centrifuged at 1000 g for 10 minutes to separate the cellular components. After centrifugation, supernatants were portioned and stored at −70°C, for no more than 2 months, pending chemokine determination. The levels of MIP-1α and MIP-1β were measured in all 42 samples using a commercial flow cytometric kit (FlowCytomix; Bender MedSystems, San Diego, California, USA) on the flow cytofluorimeter (XL-MCL; Beckman Coulter, Brea, California, USA), which was connected with BMS FlowCytomix Pro 2.2 software, according to the manufacturer’s instructions, and the concentrations of these chemokines were expressed in picograms per milliliter (pg/mL). The sensitivity of detection and standard range (lower and upper limits) were as follows: 1.0 pg/mL for MIP-1α (13.7-10 000 pg/mL), 1.0 pg/mL for MIP-1β (4.0-3000 pg/mL). The samples, analyzed in a single session, were performed in duplicate and the average value was calculated. Reproducibility within the assay was evaluated in independent experiments, as follows, for chemokine determination in the same sample: 5% for MIP-1α and MIP-1β. According to the producer’s declaration, overall intra-assay coefficient of variation should not exceed 10%.
Nasal Cytology The number of granulocytes was counted on nasal scraped tissue obtained from the inferior turbinate bilaterally by rhinoprobe. The cupped tip of the disposable probe was gently passed over the mucosal surface. Two or 3 short scrapes of the epithelial layer were made to obtain a sample. The specimen was spread onto a plain slide and immediately fixed for at least 1 minute in 95% ethyl alcohol and stained with May Grünwald-Giemsa. Samples were examined blindly by an experienced cytologist who was unaware of the clinical status of participants. The percentage of eosinophils was counted by microscopic cytological examination. The slides were examined under oil immersion by light microscopy at a magnification of 400×. Eosinophil counts were expressed as a percentage of cells of the granulocytic or mononuclear type, without nasal epithelial cells, at high power field, as an average of at least 10 fields observed.
Statistical Analysis Data were expressed as mean ± standard deviation (SD). A 1-way analysis of variance (ANOVA) was used to calculate the differences among the 3 groups of participants. To assess the levels of the statistical differences, we performed a post-hoc Dunn-Bonferroni correction. A P value of .05 or less was considered to be statistically significant. Correlations between the chemokine levels in nasal secretions and eosinophil counts/clinical parameters were evaluated by Pearson correlation test. The analysis was performed using SPSS software (Statistical Package for the Social Sciences, version 15.0; SPSS Inc, Chicago, Illinois, USA).
Results Participant Characteristics No statistically significant differences between nonallergic and allergic patients were found in the nasal symptom score, endoscopic score, and CT score. However, we found significantly higher concentrations of total serum IgE in atopic patients than in the nonatopic patients (mean [SD] = 287.38 [58.22] IU/mL vs 78.57 [21.46] IU/mL) (P = .008). The patients’ characteristics are presented in Table 1.
Chemokine Levels and Eosinophil Counts We detected MIP-1α in all nasal secretion samples of nonallergic nasal polyp patients. However, we could not detect this chemokine in samples of 3 allergic patients and 10 control participants. MIP-1β could not be detected in the nasal fluid of 5 nonallergic and 8 allergic patients, as well as 12 control participants. The average concentration of MIP-1α in nasal secretions was significantly higher in nonatopic (mean [SD] = 40.34 [12.32] pg/mL) and atopic nasal polyp
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Perić et al Table 1. Patient Characteristics. CRSwNP Patients No. of patients Age, mean ± SD, y Men/women Nasal symptom score, mean ± SD Nasal endoscopic score, mean ± SD Lund-Mackay CT score, mean ± SD Total serum IgE, mean ± SD, IU/mL
Nonallergic
Allergic
14 40.57 ± 10.28 8/6 10.57 ± 2.85 4.50 ± 1.25 15.57 ± 6.82 287.38 ± 58.22
14 41.43 ± 11.18 8/6 12.78 ± 3.07 5.00 ± 1.53 18.00 ± 5.30 78.57 ± 21.46
Abbreviations: CRSwNP, chronic rhinosinusitis with nasal polyps; CT, computed tomography; SD, standard deviation.
Figure 1. Concentrations of MIP-1α (A) and MIP-1β (B) in nasal secretions of nonallergic and allergic patients with chronic rhinosinusitis with nasal polyps and healthy participants.
patients (44.18 [33.34] pg/mL) compared to controls (8.44 [13.95] pg/mL) (P < .001, P < .0001, respectively). No significant difference in the MIP-1α levels was observed between nonallergic and allergic CRSwNP patients (P > .05) (Figure 1A). We found no significant differences in the concentration of MIP-1β among 3 investigation groups (6.97 [8.22] pg/mL in nonatopic, 10.64 [14.61] pg/mL in atopic, 3.34 [8.59] pg/mL in controls) (P > .05) (Figure 1B). The average eosinophil percentage observed in the nonatopic patients, atopic patients, and control participants were, respectively, 28.07 ± 8.83, 52.43 ± 12.00, and 6.43 ± 3.37. The highest eosinophil count was found in allergic CRSwNP patients with significant differences compared to nonallergic patients (P < .001) and control participants (P < .0001). Therefore, nonallergic nasal polyp patients have higher eosinophil counts in the nasal mucosa than control participants (P < .001) (Figure 2).
Correlations We found highly significant positive correlations between MIP-1α level in nasal secretions and nasal endoscopic score
(R = .977, P = .000) and CT score (R = .952, P = .000) and between MIP-1α concentration and eosinophil count (R = .912, P = .000), only in nonallergic patients. No significant correlations were found between MIP-1β concentrations in nasal secretions and clinical characteristics/eosinophil percentage in both nonatopic and atopic patients (Tables 2 and 3).
Discussion Interactions between eosinophils and structural cells such as epithelial cells and fibroblasts are important in the pathogenesis of tissue remodeling in CRSwNP.18,19 Data presented by Shun et al20 suggest that fibroblasts in the nasal mucosa may contribute to nasal polyp development by synthesizing CCL2 chemokine to promote macrophage recruitment. Further local hyperproduction of chemokines may participate in pathogenesis of nasal polyps by facilitating increased migration, accumulation, and activation of the immune cells, especially eosinophils, expressing the corresponding receptors, such as CCR1 and CCR3.21 Our results suggest a higher level of eosinophilic inflammation in
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Figure 2. Eosinophil counts in the nasal mucosa of nonallergic and allergic patients with chronic rhinosinusitis with nasal polyps and of control participants.
Table 2. Correlations Between Chemokine Levels in Nasal Secretions and Clinical Parameters/Eosinophil Counts in Nonallergic Patients.
MIP-1α R P value MIP-1β R P value
NSS
ES
LMS
.361 .284
.977a .000
.952a .000
−.078 .791
−.070 .813
−.016 .955
Eos Count .912a .000 −.079 .789
Abbreviations: Eos, eosinophil; ES, endoscopic score; LMS, Lund-Mackay score; NSS, nasal symptom score. a Correlation is significant at the .01 level (2-tailed).
Table 3. Correlations Between Chemokine Levels in Nasal Secretions and Clinical Parameters/Eosinophil Counts in Allergic Patients.
MIP-1α R P value MIP-1β R P value
NSS
ES
LMS
.301 .296
.068 .818
.124 .672
.161 .583
.084 .775
.041 .889
Eos Count .287 .319 .144 .623
Abbreviations: Eos, eosinophil; ES, endoscopic score; LMS, Lund-Mackay score; NSS, nasal symptom score.
allergic than in nonallergic CRSwNP patients. We could detect a higher concentration of MIP-1α in nasal secretions of atopic CRS patients than in nonatopic, but without
statistical significance. So, nasal mucosa in nonatopic and atopic patients has approximately the same ability for MIP1α production. However, the average nasal fluid concentration of MIP-1α correlates well with the eosinophil counts, endoscopic findings, and findings on CT scans, only in nonatopic CRS patients. This finding is not easy to explain. MIP-1α, among other chemokines (MIP-3α, RANTES), is responsible for induction of dendritic cell migration into the site of bacterial infection. Dendritic cells are the only antigen-presenting cells that are able to activate both Th1 and Th2 immune responses.22 The original sources of MIP-1α are monocytes and macrophages upon stimulation with bacterial endotoxins.5 After the process of cell activation, MIP1α is produced mainly by fibroblasts and eosinophils.5 Activated fibroblasts, important sources of many different chemokines, made up about 47% of the total cell population in nasal polyps, which is a significantly higher number than in the normal nasal mucosa.23 MIP-1α, released from fibroblasts and eosinophils, has been shown to induce several eosinophil responses, such as further eosinophil migration, activation, further cytokine and chemokine production, and release of eosinophil cationic protein.5 These findings could help to explain the highly significant correlation between MIP-1α concentrations, CRS severity, and eosinophil counts in the nasal mucosa of nonallergic CRS patients. Why did we not find a similar relationship in atopic CRSwNP patients? One previous investigation demonstrated that different cytokines in nasal fluid correlate well with different clinical parameters in nonatopic and atopic nasal polyp patients.24 These results suggest that chronic inflammation in allergic nasal polyp patients could be under the influence of other chemokines and cytokines. For example, De Corso et al7 found a high positive correlation between the CCL24 concentration in nasal secretions, degree of tissue eosinophilia, and severity of nasal symptoms in patients with eosinophilic nasal polyps. In another study, plasma CCL11 levels were positively correlated with the percentage of peripheral blood eosinophils and Lund-Mackay CT score.6 We propose that CCL3 (MIP-1α) in allergic nasal polyp patients probably is not as strong of a factor for eosinophil chemotaxis as CCL11 and CCL24. One explanation could be the fact that CCL11 and CCL24 are selective chemotactic factors for eosinophils, whereas MIP-1α is a potent chemotactic factor not only for eosinophils but also for basophils, monocytes, and macrophages.25 On the other hand, down-regulation of chemokine receptors during the migratory response is a very important mechanism for the adaptation and desensitization of cells to the inflammatory environment. The results of a study presented by Dulkys et al26 showed that Th2 cytokine interleukin-3 (IL-3) very efficiently induces downregulation of CCR3 receptors on the surface of human eosinophils. CCR3 is a key receptor for eosinophil
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Perić et al migration and accumulation in the nasal polyp tissue and the major chemokine receptor on eosinophils to bind MIP1α.21 As the production of IL-3 is higher in allergic CRS patients in comparison to nonallergic patients,27 it is possible that the process of eosinophil chemotaxis conducted by MIP-1α is particularly disturbed in allergic CRSwNP patients. MIP-1β (CCL4) is an inflammatory mediator that consists of 68% amino acids identical to MIP-1α.5 Our data showed very small production of MIP-1β in the inflamed mucosa of nonallergic and allergic CRS patients. These results suggest that MIP-1β has low importance for guiding chronic eosinophilic inflammation in both nonatopic and atopic CRS patients. It seems that differences in the other 32% of the polypeptide chain determine important differences in biological activity between MIP-1α and MIP-1β. On the other hand, it has been demonstrated that MIP-1α and MIP-1β exist in nonactive and active forms. After processing by activator molecule CD26/DPP, they become functional in their biological activities. For example, both MIP-1α and MIP-1β have been reported to have an important role in chemotaxis of coronary endothelial cells after heart attack.28 However, there are many differences regarding the eosinophil chemotactic and activator ability. The active form of MIP-1α shows these effects by binding to CCR1 and, especially, CCR3 receptors at the membrane of eosinophils and stromal endothelial cells, which is crucial for eosinophil attraction in the site of inflammation and transendothelial migration of eosinophils. After the activation of the MIP-1β molecule, the CCR3 signaling capacity and, hence, the chemotactic activity for eosinophils are lost.29,30 In conclusion, our results demonstrated that MIP-1α in nonallergic CRS patients plays a significant role in eosinophil chemotaxis and infiltration of the nasal and paranasal sinus mucosa. MIP-1α could be a reliable marker for assessing the severity of chronic eosinophilic inflammation only in nonatopic CRS patients. On the other hand, it is possible that chronic inflammation in atopic nasal polyp patients develops under the influence of other inflammatory mediators, probably selective chemoattractants such as eotaxin and Th2 cytokines such as IL-3. Our results suggest that allergic and nonallergic CRS patients with similar clinical characteristics have a significantly different eosinophil chemoattractant profile in nasal secretions, implying possible differences in the pathophysiology of the chronic inflammatory process. Further studies, conducted with a larger number of participants, are needed. Authors’ Note All the authors conceived of and planned the work that led to this article and interpreted the evidence that it presents. All the authors wrote this article, took part in the revision process, and approved the final version. The content of this article has not been published or submitted for publication elsewhere.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.
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research-article2014
AORXXX10.1177/0003489414554944Annals of Otology, Rhinology & LaryngologyPerić et al
Article
Macrophage Inflammatory Protein-1 Production and Eosinophil Infiltration in Chronic Rhinosinusitis With Nasal Polyps
Annals of Otology, Rhinology & Laryngology 2015, Vol. 124(4) 266–272 © The Author(s) 2014 Reprints and permissions: sagepub.com/journalsPermissions.nav DOI: 10.1177/0003489414554944 aor.sagepub.com
Aleksandar Perić, MD, PhD1, Nenad Baletić, MD, PhD1, Jelena Sotirović, MD1, and Cveta Špadijer-Mirković, MD2
Abstract Objective: Eosinophil recruitment to the nasal mucosa involves a number of chemokines. The aim of this study was to evaluate nasal secretion levels of macrophage inflammatory protein-1 alpha (MIP-1α) and MIP-1β and to correlate these levels with clinical characteristics and degree of eosinophilia in nonallergic and allergic patients with nasal polyposis (NP). Methods: Fourteen nonatopic and 14 atopic patients with NP were recruited for this cross-sectional study. Fourteen healthy subjects were included as controls. The concentrations of MIP-1α and MIP-1β in nasal secretions were measured by flow cytometry. Eosinophil counts were performed by cytological examination of the scraped nasal mucosa. We scored each of the 28 patients according to the nasal symptom score, endoscopic score, and computed tomography (CT) score. Results: We found significantly higher concentrations of MIP-1α in nasal fluid of nonallergic and allergic NP patients compared to control subjects. In nonallergic patients, we found positive correlations between MIP-1α levels and endoscopic score, CT score, and the percentage of eosinophils. Conclusion: MIP-1α may play a role in eosinophil recruitment in NP. Our results suggest that the measurement of MIP-1α in nasal secretions could be useful in evaluating the degree of eosinophil inflammation and severity of disease in nonallergic patients. Keywords chronic rhinosinusitis, nasal polyps, nasal secretions, eosinophils, chemokines, MIP-1
Introduction Chronic rhinosinusitis (CRS) is a multifactorial inflammatory disease of the nasal and paranasal sinus mucosa, and it can be divided into polypous and nonpolypous forms. Clinical and experimental studies1,2 indicate that nasal polyp growth is conducted by interactions between respiratory epithelium, lamina propria, and inflammatory cells. It may be initiated by both infectious and noninfectious inflammation. The main histological characteristic of chronic rhinosinusitis with nasal polyps (CRSwNP) is subepithelial and epithelial infiltration by eosinophils.3 Several members of the CC chemokine (C-C ligand [CCL]) subfamily, including CCL5 (regulation on activation normal T cell expressed and secreted [RANTES]), CCL7 (monocyte chemoattractant protein-3 [MCP-3]), CCL11 (eotaxin-1), CCL24 (eotaxin-2), and others, play important roles in eosinophil chemotaxis.4 One of these CC chemoattractants, macrophage inflammatory protein-1 (MIP-1), was isolated in 1988 from the supernatant of bacterial endotoxinstimulated murine macrophages. Further biochemical separation and characterization of the protein doublet yielded 2
distinct but highly related proteins, MIP-1α and MIP-1β, that shared 68% identical amino acids.5 Primary sources of these proteoglycan-binding proteins are monocytes, macrophages, and other antigen-presenting cells. After the immunological activation of other immunocompetent cells, MIP-1α and MIP-1β were found to be produced mainly by activated eosinophils and fibroblasts.5 This further MIP-1α/ MIP-1β production could be very important for further eosinophil accumulation in the nasal mucosa and prolongation of chronic inflammation. The concentrations of RANTES, eotaxin-1, and eotaxin-2 in body fluids were associated with the clinical severity of CRS.6,7 However, 1
Department of Otorhinolaryngology, Faculty of Medicine, Military Medical Academy, Belgrade, Serbia 2 Department of Otorhinolaryngology, Health Centre Kosovska Mitrovica, Serbia Corresponding Author: Aleksandar Perić, MD, PhD, Department of Otorhinolaryngology, Rhinology Unit, Faculty of Medicine, Military Medical Academy, Crnotravska 17, 11040 Belgrade, Serbia. Email: [email protected]
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Perić et al the exact role of MIP-1 in sinonasal inflammation has not been fully explored. Previous biochemical and cytological investigations showed that contents of nasal discharge reflect the inflammatory status of the nasal mucosa and the evolution of mucosal diseases.8 The relationship between CRS and allergic rhinitis is controversial. Allergic mucosal edema causes ostial obstruction that interferes with drainage and ventilation from the sinuses, promoting mucus accumulation, serum transduction, decreased oxygenation within the sinuses, and therefore, impaired ciliary movement and bacterial growth.9 The findings presented by Kirtsreesakul and Ruttanaphol9 could suggest an association between allergic rhinitis and rhinosinusitis via IgE mediated hypersensitivity.9 On the other hand, Görgülü et al10 showed no direct relationship between allergy and CRSwNP. Therefore, another study demonstrated clear differences between nonatopic and atopic nasal polyp patients regarding the inflammatory mediator profile in nasal secretions.11 The aim of this study was to evaluate nasal secretion levels of MIP-1α and MIP-1β and to investigate whether or not these concentrations correlate with severity of disease and degree of tissue eosinophilia in CRSwNP patients. The reason for this investigation was also to assess if atopic status of CRS patients has influence on MIP-1α/MIP-1β production and on their chemoattractant activities.
Methods Participants Twenty-eight (n = 28) patients with a diagnosis of CRSwNP were selected for participation in this study. Fourteen (n = 14) patients (8 men and 6 women, mean [SD] age = 40.57 [10.28] years) were nonatopic. The other 14 patients (8 men and 6 women, mean [SD] age = 41.43 [11.18] years) were atopic. This cross-sectional study was performed in accordance with the Declaration of Helsinki. The protocol and methods of the study were approved by the ethics committee of our institution. A written informed consent was obtained from all patients. This study was performed in the rhinology and allergy unit of the department of otorhinolaryngology, Military Medical Academy, Belgrade, Serbia, between September 2012 and December 2013. The diagnosis of CRSwNP was based on guidelines established by the Rhinosinusitis Task Force and endorsed and updated in 2007 by the American Academy of Otolaryngology–Head and Neck Surgery.12,13 The presence of bilateral polyps in the nasal cavities on endoscopic examination and the presence of bilateral areas of opacification in the ethmoidal labyrinths on computed tomography (CT) were the criteria for inclusion in this investigation. Exclusion criteria were the presence of choanal polyps, bronchial asthma, aspirin sensitivity, and systemic diseases affecting the nose (sarcoidosis, primary
ciliary dyskinesia, Wegener’s granulomatosis, cystic fibrosis, and Churg-Strauss syndrome). Also, patients with a history of cigarette smoking and previous paranasal sinus surgery were excluded. None of the patients had any acute upper and lower respiratory tract infections or use of antibiotics, antihistamines, or systemic or topical corticosteroids within 3 weeks before the beginning of this investigation. Fourteen healthy participants, without symptoms, medical history, and endoscopic findings of nasal/paranasal sinus inflammation, were included as controls. The mean [SD] age in the control group (9 male and 5 female participants) was 41.36 [10.05] years.
Allergy Determination The atopic status was evaluated at the beginning of the study by an allergist on the basis of clinical criteria, medical history of allergic rhinitis, positive skin-prick tests, and positive serologic test. Skin-prick tests were performed on the volar part of the forearm with a standard battery of common aeroallergens: birch, timothy, mugwort (Artemisia vulgaris), dog, cat, horse, mite (Dermatophagoides farinae, Dermatophagoides pteronyssinus), and molds (Alternaria alternata, Aspergillus fumigatus, Cladosporium herbarum, Olea europaea, Parietaria judaica, Plantago lanceolata, Platanus acerifolia). Negative (0.9% natrium-chloridum solution) and positive (1 mg/mL histamine dihydrochloride solution) control participants were also included with each skin-prick test. Reactions were read after 15 minutes, and the test was considered positive if the diameter of wheal was greater than 3 mm with respect to the negative control. Total serum IgE level was measured by an ELISA kit (Elitech Diagnostics, Salon-de-Provence, France) and an ELISA reader (Spectra III, Linz, Austria). Venous blood was collected and centrifuged, and the serum stored at −70°C until testing. Participants were considered allergic if they had a serum IgE level > 100 IU/mL.
Clinical Evaluation The same rhinologist examined all patients. All CRSwNP patients were asked to assess their symptoms (nasal obstruction, rhinorrhoea, hyposmia, sneezing, and itching). The symptoms were scored from 0 to 3 (0 for no symptoms, 1 for mild symptoms, 2 for moderate symptoms, and 3 for severe symptoms), resulting in a maximum nasal symptom score of 15, as previously described.14 Endoscopic examination was performed in all CRS patients in a sitting position with a rigid endoscope (0 and 30 degrees; Storz, Tuttlingen, Germany). The nasal polyp size scores were made based on the endoscopic findings, according to Lildholdt et al15: 0 = no polyposis; 1 = mild polyposis (small polyps that do not reach the upper edge of the inferior turbinate); 2 = moderate polyposis (medium-size polyps
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between the upper and lower edges of the inferior turbinate); 3 = severe polyposis (large polyps that reach the lower edge of the inferior turbinate). The maximum endoscopic score is 6, bilaterally. The findings from CT scans were evaluated according to the Lund-Mackay score.16 The mucosal abnormalities were graded as 0 (no abnormality), 1 (partial opacification), or 2 (total opacification) of the frontal, maxillary, anterior ethmoid, posterior ethmoid, and sphenoid sinus, bilaterally. The ostiomeatal complexes were scored bilaterally as 0 (not occluded) or 2 (occluded). The maximal CT grading score is 24.
Sampling of Nasal Secretions and Chemokine Measurement Nasal secretion samples were collected from nasal cavities of all 42 participants—28 CRS patients (14 nonallergic and 14 allergic) and 14 healthy participants—using a modified absorption technique with cotton-wool sticks (length 10 mm, diameter 4 mm; Institute of Virology, Vaccines and Sera, Torlak, Belgrade, Serbia), which were inserted for 5 minutes by bayonette forceps into the anterior part of the middle meatus, under endoscopic guidance, as previously described.11,17 All samples were placed in a 2-mL Eppendorf tube containing 1 mL of transfer medium (phosphate-buffered saline with gentamicin 50 μg/mL, penicillin G 340 IU/mL, fungizone 500 μg/mL) for 30 minutes to allow the diffusion of chemokines into the medium and then stored at 4°C for a maximum of 2 hours until processed. Nasal fluid was centrifuged at 1000 g for 10 minutes to separate the cellular components. After centrifugation, supernatants were portioned and stored at −70°C, for no more than 2 months, pending chemokine determination. The levels of MIP-1α and MIP-1β were measured in all 42 samples using a commercial flow cytometric kit (FlowCytomix; Bender MedSystems, San Diego, California, USA) on the flow cytofluorimeter (XL-MCL; Beckman Coulter, Brea, California, USA), which was connected with BMS FlowCytomix Pro 2.2 software, according to the manufacturer’s instructions, and the concentrations of these chemokines were expressed in picograms per milliliter (pg/mL). The sensitivity of detection and standard range (lower and upper limits) were as follows: 1.0 pg/mL for MIP-1α (13.7-10 000 pg/mL), 1.0 pg/mL for MIP-1β (4.0-3000 pg/mL). The samples, analyzed in a single session, were performed in duplicate and the average value was calculated. Reproducibility within the assay was evaluated in independent experiments, as follows, for chemokine determination in the same sample: 5% for MIP-1α and MIP-1β. According to the producer’s declaration, overall intra-assay coefficient of variation should not exceed 10%.
Nasal Cytology The number of granulocytes was counted on nasal scraped tissue obtained from the inferior turbinate bilaterally by rhinoprobe. The cupped tip of the disposable probe was gently passed over the mucosal surface. Two or 3 short scrapes of the epithelial layer were made to obtain a sample. The specimen was spread onto a plain slide and immediately fixed for at least 1 minute in 95% ethyl alcohol and stained with May Grünwald-Giemsa. Samples were examined blindly by an experienced cytologist who was unaware of the clinical status of participants. The percentage of eosinophils was counted by microscopic cytological examination. The slides were examined under oil immersion by light microscopy at a magnification of 400×. Eosinophil counts were expressed as a percentage of cells of the granulocytic or mononuclear type, without nasal epithelial cells, at high power field, as an average of at least 10 fields observed.
Statistical Analysis Data were expressed as mean ± standard deviation (SD). A 1-way analysis of variance (ANOVA) was used to calculate the differences among the 3 groups of participants. To assess the levels of the statistical differences, we performed a post-hoc Dunn-Bonferroni correction. A P value of .05 or less was considered to be statistically significant. Correlations between the chemokine levels in nasal secretions and eosinophil counts/clinical parameters were evaluated by Pearson correlation test. The analysis was performed using SPSS software (Statistical Package for the Social Sciences, version 15.0; SPSS Inc, Chicago, Illinois, USA).
Results Participant Characteristics No statistically significant differences between nonallergic and allergic patients were found in the nasal symptom score, endoscopic score, and CT score. However, we found significantly higher concentrations of total serum IgE in atopic patients than in the nonatopic patients (mean [SD] = 287.38 [58.22] IU/mL vs 78.57 [21.46] IU/mL) (P = .008). The patients’ characteristics are presented in Table 1.
Chemokine Levels and Eosinophil Counts We detected MIP-1α in all nasal secretion samples of nonallergic nasal polyp patients. However, we could not detect this chemokine in samples of 3 allergic patients and 10 control participants. MIP-1β could not be detected in the nasal fluid of 5 nonallergic and 8 allergic patients, as well as 12 control participants. The average concentration of MIP-1α in nasal secretions was significantly higher in nonatopic (mean [SD] = 40.34 [12.32] pg/mL) and atopic nasal polyp
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Perić et al Table 1. Patient Characteristics. CRSwNP Patients No. of patients Age, mean ± SD, y Men/women Nasal symptom score, mean ± SD Nasal endoscopic score, mean ± SD Lund-Mackay CT score, mean ± SD Total serum IgE, mean ± SD, IU/mL
Nonallergic
Allergic
14 40.57 ± 10.28 8/6 10.57 ± 2.85 4.50 ± 1.25 15.57 ± 6.82 287.38 ± 58.22
14 41.43 ± 11.18 8/6 12.78 ± 3.07 5.00 ± 1.53 18.00 ± 5.30 78.57 ± 21.46
Abbreviations: CRSwNP, chronic rhinosinusitis with nasal polyps; CT, computed tomography; SD, standard deviation.
Figure 1. Concentrations of MIP-1α (A) and MIP-1β (B) in nasal secretions of nonallergic and allergic patients with chronic rhinosinusitis with nasal polyps and healthy participants.
patients (44.18 [33.34] pg/mL) compared to controls (8.44 [13.95] pg/mL) (P < .001, P < .0001, respectively). No significant difference in the MIP-1α levels was observed between nonallergic and allergic CRSwNP patients (P > .05) (Figure 1A). We found no significant differences in the concentration of MIP-1β among 3 investigation groups (6.97 [8.22] pg/mL in nonatopic, 10.64 [14.61] pg/mL in atopic, 3.34 [8.59] pg/mL in controls) (P > .05) (Figure 1B). The average eosinophil percentage observed in the nonatopic patients, atopic patients, and control participants were, respectively, 28.07 ± 8.83, 52.43 ± 12.00, and 6.43 ± 3.37. The highest eosinophil count was found in allergic CRSwNP patients with significant differences compared to nonallergic patients (P < .001) and control participants (P < .0001). Therefore, nonallergic nasal polyp patients have higher eosinophil counts in the nasal mucosa than control participants (P < .001) (Figure 2).
Correlations We found highly significant positive correlations between MIP-1α level in nasal secretions and nasal endoscopic score
(R = .977, P = .000) and CT score (R = .952, P = .000) and between MIP-1α concentration and eosinophil count (R = .912, P = .000), only in nonallergic patients. No significant correlations were found between MIP-1β concentrations in nasal secretions and clinical characteristics/eosinophil percentage in both nonatopic and atopic patients (Tables 2 and 3).
Discussion Interactions between eosinophils and structural cells such as epithelial cells and fibroblasts are important in the pathogenesis of tissue remodeling in CRSwNP.18,19 Data presented by Shun et al20 suggest that fibroblasts in the nasal mucosa may contribute to nasal polyp development by synthesizing CCL2 chemokine to promote macrophage recruitment. Further local hyperproduction of chemokines may participate in pathogenesis of nasal polyps by facilitating increased migration, accumulation, and activation of the immune cells, especially eosinophils, expressing the corresponding receptors, such as CCR1 and CCR3.21 Our results suggest a higher level of eosinophilic inflammation in
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Figure 2. Eosinophil counts in the nasal mucosa of nonallergic and allergic patients with chronic rhinosinusitis with nasal polyps and of control participants.
Table 2. Correlations Between Chemokine Levels in Nasal Secretions and Clinical Parameters/Eosinophil Counts in Nonallergic Patients.
MIP-1α R P value MIP-1β R P value
NSS
ES
LMS
.361 .284
.977a .000
.952a .000
−.078 .791
−.070 .813
−.016 .955
Eos Count .912a .000 −.079 .789
Abbreviations: Eos, eosinophil; ES, endoscopic score; LMS, Lund-Mackay score; NSS, nasal symptom score. a Correlation is significant at the .01 level (2-tailed).
Table 3. Correlations Between Chemokine Levels in Nasal Secretions and Clinical Parameters/Eosinophil Counts in Allergic Patients.
MIP-1α R P value MIP-1β R P value
NSS
ES
LMS
.301 .296
.068 .818
.124 .672
.161 .583
.084 .775
.041 .889
Eos Count .287 .319 .144 .623
Abbreviations: Eos, eosinophil; ES, endoscopic score; LMS, Lund-Mackay score; NSS, nasal symptom score.
allergic than in nonallergic CRSwNP patients. We could detect a higher concentration of MIP-1α in nasal secretions of atopic CRS patients than in nonatopic, but without
statistical significance. So, nasal mucosa in nonatopic and atopic patients has approximately the same ability for MIP1α production. However, the average nasal fluid concentration of MIP-1α correlates well with the eosinophil counts, endoscopic findings, and findings on CT scans, only in nonatopic CRS patients. This finding is not easy to explain. MIP-1α, among other chemokines (MIP-3α, RANTES), is responsible for induction of dendritic cell migration into the site of bacterial infection. Dendritic cells are the only antigen-presenting cells that are able to activate both Th1 and Th2 immune responses.22 The original sources of MIP-1α are monocytes and macrophages upon stimulation with bacterial endotoxins.5 After the process of cell activation, MIP1α is produced mainly by fibroblasts and eosinophils.5 Activated fibroblasts, important sources of many different chemokines, made up about 47% of the total cell population in nasal polyps, which is a significantly higher number than in the normal nasal mucosa.23 MIP-1α, released from fibroblasts and eosinophils, has been shown to induce several eosinophil responses, such as further eosinophil migration, activation, further cytokine and chemokine production, and release of eosinophil cationic protein.5 These findings could help to explain the highly significant correlation between MIP-1α concentrations, CRS severity, and eosinophil counts in the nasal mucosa of nonallergic CRS patients. Why did we not find a similar relationship in atopic CRSwNP patients? One previous investigation demonstrated that different cytokines in nasal fluid correlate well with different clinical parameters in nonatopic and atopic nasal polyp patients.24 These results suggest that chronic inflammation in allergic nasal polyp patients could be under the influence of other chemokines and cytokines. For example, De Corso et al7 found a high positive correlation between the CCL24 concentration in nasal secretions, degree of tissue eosinophilia, and severity of nasal symptoms in patients with eosinophilic nasal polyps. In another study, plasma CCL11 levels were positively correlated with the percentage of peripheral blood eosinophils and Lund-Mackay CT score.6 We propose that CCL3 (MIP-1α) in allergic nasal polyp patients probably is not as strong of a factor for eosinophil chemotaxis as CCL11 and CCL24. One explanation could be the fact that CCL11 and CCL24 are selective chemotactic factors for eosinophils, whereas MIP-1α is a potent chemotactic factor not only for eosinophils but also for basophils, monocytes, and macrophages.25 On the other hand, down-regulation of chemokine receptors during the migratory response is a very important mechanism for the adaptation and desensitization of cells to the inflammatory environment. The results of a study presented by Dulkys et al26 showed that Th2 cytokine interleukin-3 (IL-3) very efficiently induces downregulation of CCR3 receptors on the surface of human eosinophils. CCR3 is a key receptor for eosinophil
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Perić et al migration and accumulation in the nasal polyp tissue and the major chemokine receptor on eosinophils to bind MIP1α.21 As the production of IL-3 is higher in allergic CRS patients in comparison to nonallergic patients,27 it is possible that the process of eosinophil chemotaxis conducted by MIP-1α is particularly disturbed in allergic CRSwNP patients. MIP-1β (CCL4) is an inflammatory mediator that consists of 68% amino acids identical to MIP-1α.5 Our data showed very small production of MIP-1β in the inflamed mucosa of nonallergic and allergic CRS patients. These results suggest that MIP-1β has low importance for guiding chronic eosinophilic inflammation in both nonatopic and atopic CRS patients. It seems that differences in the other 32% of the polypeptide chain determine important differences in biological activity between MIP-1α and MIP-1β. On the other hand, it has been demonstrated that MIP-1α and MIP-1β exist in nonactive and active forms. After processing by activator molecule CD26/DPP, they become functional in their biological activities. For example, both MIP-1α and MIP-1β have been reported to have an important role in chemotaxis of coronary endothelial cells after heart attack.28 However, there are many differences regarding the eosinophil chemotactic and activator ability. The active form of MIP-1α shows these effects by binding to CCR1 and, especially, CCR3 receptors at the membrane of eosinophils and stromal endothelial cells, which is crucial for eosinophil attraction in the site of inflammation and transendothelial migration of eosinophils. After the activation of the MIP-1β molecule, the CCR3 signaling capacity and, hence, the chemotactic activity for eosinophils are lost.29,30 In conclusion, our results demonstrated that MIP-1α in nonallergic CRS patients plays a significant role in eosinophil chemotaxis and infiltration of the nasal and paranasal sinus mucosa. MIP-1α could be a reliable marker for assessing the severity of chronic eosinophilic inflammation only in nonatopic CRS patients. On the other hand, it is possible that chronic inflammation in atopic nasal polyp patients develops under the influence of other inflammatory mediators, probably selective chemoattractants such as eotaxin and Th2 cytokines such as IL-3. Our results suggest that allergic and nonallergic CRS patients with similar clinical characteristics have a significantly different eosinophil chemoattractant profile in nasal secretions, implying possible differences in the pathophysiology of the chronic inflammatory process. Further studies, conducted with a larger number of participants, are needed. Authors’ Note All the authors conceived of and planned the work that led to this article and interpreted the evidence that it presents. All the authors wrote this article, took part in the revision process, and approved the final version. The content of this article has not been published or submitted for publication elsewhere.
Declaration of Conflicting Interests The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.
Funding The author(s) received no financial support for the research, authorship, and/or publication of this article.
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