Quantitative Analysis of Eotaxin and RANTES Messenger RNA in Nasal Polyps: Association of Tissue and Nasal Eosinophils

Authors

  • Seung-Heon Shin MD,

    Corresponding author
    1. Department of Otolaryngology, School of Medicine, Kyungpook National University, Taegu, Korea
    • Seung-Heon Shin, MD, Department of Otolaryngology, Taegu Catholic University Hospital 3056-6 Daemyung 4 Dong, Nam-Gu, Taegu, South Korea 705-718.
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  • Jae-Yul Park MD,

    1. Department of Otolaryngology, School of Medicine, Kyungpook National University, Taegu, Korea
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  • Chang-Ho Jeon MD,

    1. Department of Clinical Pathology, School of Medicine, Kyungpook National University, Taegu, Korea
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  • Jin-Kyo Choi MD,

    1. School of Medicine, Catholic University of Taegu-Hyosung College of Medicine; and the Department of Otolaryngology, School of Medicine, Kyungpook National University, Taegu, Korea
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  • Sang-Heon Lee MD

    1. School of Medicine, Catholic University of Taegu-Hyosung College of Medicine; and the Department of Otolaryngology, School of Medicine, Kyungpook National University, Taegu, Korea
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Abstract

Objectives/Hypothesis Nasal polyps develop in the ethmoidal and middle turbinate area, often in relation to inflammatory conditions. Their exact etiology and pathogenesis are still under debate. Histologically, the polyps are infiltrated by a number of inflammatory cells, with eosinophil predominating in most specimens. This finding suggests that the nasal polyp is an inflammatory growth that is controlled by the local environment. The chemokines eotaxin and RANTES (r egulated on a ctivation n ormal T cell e xpressed and s ecreted) have been postulated to be involved in the recruitment and activation of eosinophils to certain inflamed tissues. The purpose of this study was to investigate eotaxin and RANTES mRNA expression in nasal polyps and its effect on tissue and nasal eosinophils.

Methods Nasal polyps (917 allergic and 30 nonallergic cases) were obtained from endoscopic sinus surgery, and 15 normal inferior turbinates also were taken. Immunohistochemical staining for eosinophils and quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) tests for eotaxin and RANTES mRNA expression were performed, and the concentration of nasal eosinophil cationic protein (ECP) was measured.

Results The amounts of eotaxin mRNA in the allergic nasal polyps were 11.4 times higher and the levels in the nonallergic polyps were 6.4 times higher than in the normal inferior turbinate. However, the RANTES mRNA expression did not show any differences among the three groups. Tissue eosinophilia and nasal ECP levels were significantly correlated with eotaxin mRNA level but not with RANTES mRNA expression.

Conclusion Nasal polyp eosinophilic infiltration and activation correlate mainly with increased eotaxin gene expression rather than with RANTES expression.

INTRODUCTION

Nasal polyps develop from the respiratory tract mucosa of the ethmoidal and middle turbinate area. Inflammatory conditions of the nasal mucosa may play an important role in the development of polyps, but the etiology and pathogenesis are not yet fully understood. Histologically, the polyps are different from normal nasal mucosa, consisting of respiratory tract epithelium covering very edematous stroma infiltrated by a number of inflammatory cells such as basophils, mast cells, and moderate to high numbers of eosinophils. These findings suggest that the nasal polyp is an inflammatory growth that is controlled by the local microenvironment. 1,2

Nasal polyps, bronchial asthma, parasitic infection, and several other inflammatory diseases are associated with increased number of eosinophils. These cells release inflammatory products from their granules, including major basic protein (MBP), eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), and leukotrienes. The products damage the epithelium of the upper and lower respiratory tract. 3 The accumulation of eosinophils in a tissue site is associated with a number of events, including production of mature cells from bone marrow, adhesion and migration through the vascular endothelium, and an increase in survival. Interleukin (IL)-3, IL-5, and granulocyte-macrophage colony-stimulating factor (GM-CSF) play significant roles in the promotion of survival and activation of mature eosinophils. 4

The accumulation of leukocytes in tissues requires at least three stages of leukocyte and endothelial interaction. Initially, the circulating leukocytes are captured and roll on the endothelial cells, a process mediated by selectins. The second step is tight binding and activation by chemoattractants. Third, the leukocyte migrates along a concentration gradient of chemokines to the interstitium. 5,6

The chemokines are a family of 8- to 10-kD soluble proteins having 20% to 90% amino acid homology. Four classes of chemokines have been defined by the arrangement of conserved cysteine residues of the mature proteins: the residues CXC, CC, C, and CX3C. The main function of chemokines appears to be related to the selective activation and recruitment of particular leukocyte subsets, but a number of different roles have been ascribed. 7,8 The members of the CXC chemokine family, such as IL-8 and neutrophil-activation protein activate, predominantly, neutrophils, whereas the CC chemokine family members, such as macrophage inflammatory protein (MIP)-1α, monocyte chemoattractant proteins 2 and 3 (MCP-2 and MCP-3), RANTES (r egulated on a ctivation n ormal T cell e xpressed and s ecreted), and eotaxin generally attract other leukocytes such as monocytes, T cells, basophils, and eosinophils. RANTES, eotaxin, and MCP-3 activate, predominantly, eosinophils. 9–11 In the present study, we assessed expression of eotaxin and RANTES messenger RNA (mRNA) in human nasal polyps and also sought a correlation between the expression of chemokines and tissue eosinophilia and its activity.

MATERIALS AND METHODS

Patients

Forty-seven patients (age range, 18–67 y; mean age, 39.7 y; SD = 14.7 y) undergoing endoscopic sinus surgery for bilateral nasal polyps were studied. On the basis of the results of allergy skin testing, these patients were divided into allergic (n = 17) and nonallergic (n = 30) groups. A positive skin prick test result was defined as a wheal that equals 3 mm in diameter compared with the negative control at 15 minutes, in response to one or more extracts of 30 aeroallergens. Patients were excluded if they had received systemic and topical steroids, antihistamines, or antibiotics during the 4 weeks preceding the study. Fifteen control specimens were obtained from the inferior turbinates of patients who did not have a history of allergy or infection.

Quantitative Reverse Transcriptase–Polymerase Chain Reaction Testing of Eotaxin and RANTES Messenger RNA in Nasal Polyps

Frozen tissues of nasal polyps were weighed, and total RNA was isolated according to the manual of the SV Total RNA Isolation Kit (Promega Co., Madison, WI). We amplified complementary DNA (cDNA) using the reverse transcription system (Promega) with 9.5 μL of total RNA. A total volume of 20 μL of reverse transcriptase–polymerase chain reaction (RT-PCR) test components were reacted for 10 minutes at 25°C, 60 minutes at 42°C, and 5 minutes at 99°C.

From the amplified cDNA, we did quantitative PCR of reduced glyceraldehyde-phosphate dehydrogenase (GAPDH), eotaxin, and RANTES in a 96-well plate using SYBR Green PCR core kit (PE Applied Biosystems, Foster City, CA) with the GeneAmp 5700 system (PE Applied Biosystems). The reagents used for the PCR were as follows: Ampli Taq Gold (PE Biosystems, Foster City, CA) DNA polymerase 1 U/μL, 0.25 μL; deoxyribonucleoside triphosphate (dNTP) mix (2.5 mmol/L of deoxyadenosine triphosphate [dATP], deoxycytidine triphosphate [dCTP], and deoxyguanosine triphosphate [dGTP]) and 5.0 mmol/L of deoxyuridine triphosphate [dUTP], 4 μL; AmpErase (PE Biosystems) uracil-N-glycosylase 1 U/μL, 0.5 μL; 10X SYBR green buffer, 5 μL; 25 mmol/L MgCL2 solution, 6 μL; cDNA, 4 μL; primers, 2 μL; and distilled water, 28.25 μL.

To activate the uracil-N-glycosylase and Ampli Taq Gold DNA polymerase, we incubated the PCR mixture at 50°C for 2 minutes and then at 95°C for 10 minutes. After that, we amplified the three targets at the same time using 40 cycles of denaturation (95°C for 15 s) and annealing (60°C for 1 min). The primer sequences and amplified products were as follows:

  • GAPDH F: 5′-GTC AAC GGA TTT GGT CTG TAT T-3′.

  • GAPDH R: 5′-AGT CTT CTG GGT GGC AGT GAT-3′, 560 base pairs (bp).

  • Eotaxin F: 5′-TTA TGG CTT TGG AGT TGG AGA-3′

  • Eotaxin R: 5′-AGA GGT TGA GGT TTC GGT ATT-3′, 297 bp.

  • RANTES F: 5′-ATA TTC CTC GGA CAC CAC AC-3′.

  • RANTES R: 5′-CAC TCC AGC CTG GGG AAG G-3′, 370 bp.

As SYBR green combined with the target DNAs, the GeneAmp 5700 system could detect the fluorescence emission in a closed-well reaction plate after each cycle. The software monitored the data at every cycle throughout the PCR and calculated the threshold cycle (CT) where the amplification plot crossed a defined fluorescence threshold. For the quantitation of the PCR products, we used the relative quantitative methods with Mycobacterium tuberculosis DNA as standards. We measured M tuberculosis DNA concentration by TKO 100 fluorometer (Hoefer Scientific Instruments, San Francisco, CA) and made DNA concentration standards of 10 pg, 5.5 pg, 1 pg, 100 fg, 50 fg, 10 fg, and 6.13 fg. Then we amplified these standards with the specimens for GAPDH, eotaxin, and RANTES in the same plate and prepared the standard curve with the log input amount as the Y values and CT as the X values. According to the curve, we calculated the relative concentration of GAPDH, eotaxin, and RANTES. The concentrations of eotaxin and RANTES were divided by that of GAPDH, and the normalized ratios were divided by the normalized ratios of controls. In this way, we could estimate the relative expression of eotaxin and RANTES mRNA compared with normal controls.

Immunohistochemical Study for Eosinophil Major Basic Protein in Nasal Polyps

The tissue biopsies were frozen in liquid nitrogen and stored at −70°C until used. Cryostat sections of 6 to 8 μm were prepared and treated with 1% H2O2 for 30 minutes to block endogenous peroxidase. The slides were washed three times in phosphate-buffered saline (PBS). Endogenous biotin was quenched by pretreatment of the sections with an avidin-biotin blocking kit (Vector, Burlingame, CA) for 20 minutes at room temperature. The slides were incubated with 1:20 anti-BMK13 (Sanbio, Uden, The Netherlands) for 10 minutes at room temperature, washed three times in 0.05 mol/L Tris-buffered saline (TBS), and incubated with biotin-labeled secondary antibodies for 10 minutes at room temperature. After three washings in TBS, the slides were incubated with streptavidin peroxidase (Histostain-SP kit, Zymed, South San Francisco, CA), and counterstaining was performed with hematoxylin. Tonsil sections were used as positive controls.

Counts of positive cells were made on all sections in the subepithelial area in four fields at original magnification × 400. Two investigators who did not know the clinical status of the patients and controls counted all specimens independently, and the results were averaged. To determine the activity of eosinophils, extracellular deposition of MBP granules was measured. The extent of extracellular deposition was graded from 0 to 4 (0, none; 1, <10% of section area; 2, 10%–50% of section area; 3, 50%–70% of section area; 4, >75% of section area). 12

Detection of Eosinophil Cationic Protein in Nasal Secretions

Nasal secretions were collected by repeated aspiration from the middle and inferior meatus into a preweighed plastic sampling tube. This was immediately followed by aspiration of a known volume of 0.01 mol dithiothreitol (DTT) in PBS. The samples were cooled on ice immediately after sampling and centrifuged at 1000 g at 4°C for 15 minutes. The supernatant liquids were stored at −70°C until used. The ECP concentrations were measured with the Pharmacia CAP system (Pharmacia & Upjohn, Stockholm, Sweden).

Statistical Analysis

The expression of eotaxin, RANTES, MBP-positive cells, and nasal ECP concentrations in the three groups was compared using one-way ANOVA with subsequent post hoc Scheffe testing. Correlation coefficients were obtained by Spearman's rank method for extracellular deposition of MBP granules and Pearson's rank method for MBP-positive cells and nasal ECP level. A value of P < .05 was considered significant.

RESULTS

Quantitative Reverse Transcriptase–Polymerase Chain Reaction Testing of Eotaxin and RANTES

The changes of CT value according to the input of M tuberculosis DNA are shown in Figure 1. There was good linearity from 10,000 fg to 10 fg (P > .01).

Figure Fig. 1..

Correlation between Mycobacterium tuberculosis DNA concentration and threshold cycle value.

Both allergic and nonallergic polyps had increased expression of eotaxin and RANTES mRNA compared with normal inferior turbinates. The expression of eotaxin mRNA in allergic polyps was significantly higher than in normal control (P < .05), but this was not true of the nonallergic polyps. In nasal polyps, the RANTES mRNA level was 1.3 times that in inferior turbinates, but the difference was not statistically significant (P > .05) (Fig. 2).

Figure Fig. 2..

Eotaxin and RANTES messenger RNA (mRNA) expression (A) in nasal polyps of allergic and nonallergic patients relative to their expression in normal controls, (B) immunohistochemical staining of major basic protein (MBP) (original magnification × 400), MBP-positive cell count and extracellular deposit of MBP granules, (C) nasal eosinophil cationic protein (ECP) level (ng/mL). Mean values are represented by horizontal bars. AP = allergic polyp; NAP = nonallergic polyp; NC = normal control.

Immunohistochemical Results for Eosinophil Concentration and Correlation With Chemokines

The number of BMK13-positive eosinophils per high-power field was significantly elevated in nasal polyps compared with inferior turbinate. Extensive extracellular MBP deposition was seen in nasal polyps but not in inferior turbinates (P < .05) (Fig. 2).

The eotaxin mRNA expression from nasal polyps correlated with the number of BMK13-positive eosinophils (γ = 0.05;P < .01) and extracellular MBP granule deposition (γ = 0.60;P < .01). However, the RANTES mRNA did not correlate with either (Fig. 3).

Figure Fig. 3..

Correlation between eotaxin mRNA expression and tissue eosinophilia and nasal ECP levels.

Nasal Eosinophil Cationic Protein Concentration and Correlation With Chemokines

The ECP concentrations in the nasal secretion of polyps (453.7ng/mL in allergic, 293.2 ng/mL in nonallergic specimens) were higher than in controls (P < .05). There was no statistically significant difference between the polyp groups (Fig. 2). Eotaxin mRNA expression was significantly correlated with the concentration of nasal ECP (γ = 0.38;P = .05) but not with the RANTES mRNA expression (γ = 0.03;P = .85)(Fig. 3).

DISCUSSION

Nasal polyps can be defined as a chronic inflammatory disease of the paranasal sinus and nasal mucosa, leading to a protrusion of benign edematous polyps from the meatus into the nasal cavity. It is likely that nasal polyps involve multiple factors that interact to set forth a cascade of inflammatory responses that culminate in polypoid nasal growth. To understand the inflammatory immunological mechanisms, the role of specific cytokines, which function as mediators of many of the intercellular interactions and recruitment of cells, should be investigated.

The development of nasal polyp eosinophilia depends on the presence of selective priming cytokines such as IL-3, IL-5, GM-CSF, and CC chemokines such as RANTES, eotaxin, and MCP-3. 9,10 RANTES is released by macrophages, platelets, fibroblasts, vascular endothelial cells, lymphocytes, and epithelial cell lines. It has a potent chemotactic activity for eosinophils and T lymphocytes. 9,10 RANTES is a strong inducer of eosinophil transendothelial migration, and this effect is enhanced by pretreatment of eosinophils with IL-5. 13 RANTES also induces the release of ECP and superoxide anions from eosinophils, so it has a dual effect on eosinophils: activation and recruitment. 9 The effect of RANTES on eosinophil activation is as strong as that of platelet activating factor, but RANTES does not appear to influence eosinophil survival. The RANTES mRNA expression in nasal polyps was 60% to 97%, and there was no significant difference between chronic sinusitis and nasal polyp tissues. It induces increased recruitment and activation of eosinophils. 14,15 The present study showed that entire nasal polyps and inferior turbinates expressed RANTES. This difference from previous findings might reflect our use of fluorescent method with SYBR green to measure the PCR product, whereas other investigators used an electrophoresis method that cannot detect very small amounts of the molecules (less than 1 ng/mL). Bartels et al. 16 previously reported that nonatopic and atopic nasal polyps express more RANTES mRNA than normal nasal mucosa, but in the present study, RANTES mRNA expression was 1.3 times higher in nasal polyps than in normal controls. Nevertheless, there was no statistical significance, and no correlation was seen between RANTES and BMK13-positive eosinophils, extracellular deposition of MBP granules, and nasal ECP concentration. Moreover, there were no significant differences in RANTES protein expression among the three groups, as judged by immunohistochemical staging for RANTES (data not shown, R&D Systems, Minneapolis, MN). Therefore we suggest that RANTES does not play a critical role in tissue eosinophilia and activation in nasal polyps. These results are somewhat different from those of previous studies, 14,15 but our results can be explained by the increased eosinophil adhesion to nasal epithelial cells. This could cause signal transfer from eosinophils to inhibit RANTES production as a negative feedback mechanism. As an alternative, RANTES may not be critical for the recruitment of eosinophils. 17

Eotaxin is 8 kD and shows 34.2% amino acid sequence identity with RANTES. It acts highly selectively on eosinophils and is upregulated by cytokines such as IL-3, IL-4, interferon-γ (INF-γ), and tumor necrosis factor-α (TNF-α), which have been associated with allergic and nonallergic eosinophilic inflammation. 6,18 Most eotaxin appears to be derived from macrophages, eosinophils, and T lymphocytes within the nasal submucosa. 19 In this study, eotaxin mRNA expression was significantly higher in nasal polyps (11.4 times in allergic and 6.4 times in nonallergic specimens) than in normal inferior turbinates, and there were no significant differences between polyps. This result agrees with those reported by other authors. 16,19 For example, Minshall et al. 19 reported that eotaxin protein was significantly increased in both allergic and nonallergic sinusitis. This increase might be related to several cytokines that have been proposed to enhance eotaxin production. Cytokine expression in allergic sinusitis resembles a helper T cell type 2 (Th2) type of response with increased secretion of GM-CSF, IL-3, IL-4, and IL-5. In nonallergic sinusitis, eotaxin production is associated with the helper T cell type 1 (Th1) type of cytokines such as GM-CSF, IL-3, TNF-α, and INF-γ. 18 The distinct cytokine pathways would operate to enhance eotaxin expression in allergic and nonallergic reactions. The CC chemokines, together with eosinophil-active cytokines, may contribute nasal mucosal accumulation of eosinophils in both allergic and nonallergic nasal polyps. Eotaxin is as potent as RANTES in eosinophil chemotaxis, but in our study, eotaxin was more significantly correlated with eosinophil recruitment and activation. 20 These finding suggest that eotaxin plays an important role in allergic and nonallergic eosinophilic inflammation in nasal polyps.

CONCLUSION

Our study of eotaxin and RANTES mRNA expression and its relation to eosinophils demonstrated that eosinophilic infiltration and activation in nasal polyps correlates mainly with eotaxin mRNA rather than RANTES mRNA. Eotaxin may play a more important role than RANTES in polyp formation.

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