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Eotaxin, RANTES and tumor necrosis factor alpha levels in allergic rhinitis
Egyptian Journal of Ear, Nose, Throat and Allied Sciences (2011) 12, 33–37
Egyptian Society of Ear, Nose, Throat and Allied Sciences
Egyptian Journal of Ear, Nose, Throat and Allied Sciences www.esentas.org
ORIGINAL ARTICLE
Eotaxin, RANTES and tumor necrosis factor alpha levels in allergic rhinitis Asser El Sharkawy a b
a,*
, Shawky Elmorsy a, Mohammed El-Naggar
b
Department of Otorhinolaryngology, Faculty of Medicine, Mansoura University, Mansoura, Egypt Department of Medical Microbiology & Immunology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
Received 11 December 2010; accepted 9 February 2011 Available online 23 June 2011
KEYWORDS Allergic rhinitis; Chemokines; Tumor necrosis factor alpha
Abstract Objectives: The objectives of this study were to estimate the levels of eotaxin, RANTES and tumor necrosing factor-1 in allergic rhinitis and their relation to disease severity. Study design: Prospective study. Setting: Mansoura University Hospital. Patients and methods: Twenty nine patients suffering from allergic rhinitis were included in this study (19 patients with allergic rhinitis and 10 patients control group). The patients underwent estimation of eotaxin, RANTES, TNF-1 in the nasal wash using Elisa technique. The patients were divided according to the disease severity into mild allergic rhinitis, severe allergic rhinitis and control groups. Results: The mean values of eotaxin, RANTES, TNF-1 in severe allergic rhinitis (33.6 ± 11.07 pg/ml, 72.17 ± 87.61 pg/ml, 25.47 ± 4.04 pg/ml) were statistically higher than in mild allergic rhinitis (9.80 ± 6.79 pg/ml, 10.50 ± 6.90 pg/ml, 12.99 ± 3.27 pg/ml) and the mean values of all these parameters were higher in both groups compared to control group (0.6 ± 0.69 pg/ml, 0.65 ± 0.74 pg/ml, 0.63 ± 0.54 pg/ml).
* Corresponding author. Address: ORL Department, Mansoura University Hospital, Mansoura, Egypt. Tel.: +20 102223269; fax: +20 502267016. E-mail address: [email protected] (A.El Sharkawy). 2090-0740 ª 2011 Egyptian Society of Ear, Nose, Throat and Allied Sciences. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of Egyptian Society of Ear, Nose, Throat and Allied Sciences. doi:10.1016/j.ejenta.2011.02.001
Production and hosting by Elsevier
34
A.E. Sharkawy et al. Conclusion: This study suggests the role of local chemokines in the pathogenesis of allergic rhinitis as well as their possible relation to the severity of the disease which may direct the attention to therapeutic trials against these locally produced chemokines. ª 2011 Egyptian Society of Ear, Nose, Throat and Allied Sciences. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction
2. Patients and methods
There is accumulating evidence that chemokines are involved in both the migration and the activation of eosinophil and other leukocytes during asthma responses. These chemokines include RANTES, macrophage-inflammatory protein-11 (MIP-11), monocyte-chemotactic protein-3 (MCP-3), eotaxin, and MCP-5.1 Eotaxin is the major eosinophil chemoattractant that binds with high affinity to chemokin receptor 3 (CCR3) which is expressed on cells such as eosinophils, basophils, and Th-2 lymphocytes.2 It is secreted by multiple cell types including epithelium, fibroblasts, smooth muscle cells, and eosinophils.3 Eotaxin family production by eosinophils results in the recruitment of eosinophils into the tissue by a self-amplifying process in eosinophilic chronic rhinosinusitis patients.4 All members of the Eotaxin family (Eotaxin-1, -2 and -3) are involved in the pathogenesis of nasal polyposis. The results are more likely indicative of a complex cooperation between all members of the geotaxis family than of a specific role in the development of eosinophilia and nasal polyposis.5 RANTES is a broad-acting chemokine that has an established role in the recruitment of CD4+ T cells, monocytes, and eosinophils into inflammatory sites, and is upregulated in patients with asthma. It is produced by numerous lung cell types including CD8+ T cells and bronchial epithelial cells.6 Fujikura et al.7 investigated the expression of RANTES, and eotaxin in the nasal mucosa of patients with allergic rhinitis to clarify the effect of histamine and antihistamine on the regulation of the expression of these Cysteine–Cysteine (CC) chemokines. They concluded that histamine may induce CC chemokine production in the nasal mucosa of patients with allergic rhinitis. They suggested that the regulation of histamine-CC chemokine interaction could lead to new therapeutic approaches for the treatment of nasal allergy. Nasal allergen challenge induces the enhanced secretion of IL-17 and RANTES in the lower airways of nonasthmatic patients with allergic rhinitis.8 Although TNF-1 can cause the characteristic physiological phenotype of asthma, namely airway hyper responsiveness and eosinophilia, the fact that it is not a potent chemoattractant for eosinophils suggests that its effects on eosinophil recruitment are indirect.9 Lilly et al.10 demonstrates that TNF-1 and IL-1b rapidly and transiently induce the accumulation of eotaxin mRNA in bronchial epithelial cell lines and that this increase is associated with the intracellular accumulation of eotaxin protein and its secretion into the cell supernatant. TNF-1 produces a nasal inflammatory response in humans that is characterized by late phase (i.e., 24 h post challenge) neutrophil activity and plasma exudation.11 The aim of this study was to evaluate the levels of eotaxin, RANTES and tumor necrosing factor-1 in allergic rhinitis and their possible relation to disease severity.
Ethical approval for this work was obtained by the Ethical Committee Board of Faculty of Medicine, Mansoura University. This work was done in ORL department and Microbiology and immunology departments in the period between August 2007 and May 2008. The study included 29 patients (19 patients with allergic rhinitis and 10 control). The patients were selected from the out patients clinic of the ORL department Mansoura university. The diagnosis of allergic rhinitis was confirmed by clinical history, clinical examination, and positive skin test for certain allergen and elevated total serum IgE. The patients were classified into three groups: group I (n = 10) patients with severe allergic symptoms, group II (n = 9) patients with mild allergic symptoms based on visual analogue scale (VAS).12 The previous groups were compared with nonsmoking subjects with no positive skin-prick response to common aeroallergens or history of respiratory disease acted as a control group III (n = 10). 2.1. Nasal wash samples The nasal cavity was washed with 10 ml of physiological saline warmed at 37 C by reciprocating the piston of the syringe 10 times on each side. Lavage fluid was centrifuged for 20 min at 400·g. The supernatant was stored frozen at 30 C until assayed.8 2.2. Measurement of eotaxin Eotaxin was measured in nasal wash using ELISA technique. The BioSource human Eotaxin (hEotaxin) ELISA was used (BioSource, Nivelles, Belgium). Fifty microliters of the standard diluent buffer were added to the wells reserved for samples and zero well. Fifty microliters of standards, samples or controls were added to the appropriate microtiter wells. One hundred microliters of biotinylated anti-eotaxin solution were pipette into each well. After incubation for 2 h, wells were aspirated and washed. One hundred microliters Streptavidin-HRP working solution were added to each well and incubated for 30 min and thereafter, wells were thoroughly aspirated and washed. One hundred microliters of stabilized chromogen were added to each well. After incubation for 30 min, 100 ll of stop solution were added to each well. Absorbance of each well was read at 450 nm. 2.3. Measurement of RANTES RANTES was measured in nasal wash and polyp samples using ELISA technique. The BioSource human RANTES (hRANTES) ELISA was used (BioSource, Nivelles, Belgium). Fifty microliters of the standard diluent buffer were added to zero wells. Fifty microliters of standards, samples or controls
Eotaxin, RANTES and tumor necrosis factor alpha levels in allergic rhinitis Table 1
35
Eotaxin, Rantes and TNF-1 and total serum IgE in different groups.
Group I Group II Group III (control group) P* P** P***
Eotaxin (pg/ml)
RANTES (pg/ml)
TNF-1 (pg/ml)
Total IgE (IU/ml)
M
M
M
SD
M
SD
4.04 3.27 0.54
626 593.9 31.5 0.803 <0.001 <0.001
281.78 286.23 11.03
33.6 9.80 0.6 <0.001 <0.001 <0.001
SD 11.07 6.79 0.69
72.17 10.50 0.65 0.04 0.019 <0.001
SD 87.61 6.90 0.74
25.47 12.99 0.63 <0.001 <0.001 <0.001
*
P (severe allergic rhinitis vs. mild allergic rhinitis). P (severe allergic rhinitis vs. control). *** P (mild allergic rhinitis vs. control). **
were added to the appropriate microtiter wells and 50 ll of biotinylated anti-RANTES solution were pipette into each well and incubated for 1 h. Wells were aspirated and washed. One hundred microliters Streptavidin-HRP working solution was added to each well. After incubation for 30 min, wells were aspirated and washed and 100 ll of stabilized chromogen were added to each well. After incubation for 30 min, 100 ll of stop solution were added to each well. Absorbance of each well was read at 450 nm. 2.4. Measurement of tumor necrosis factor-1 EASIA TM ELISA Kit (Biosource, Nivelles, Belgium) was used. Fifty microliters incubation buffers were added to all wells, 200 ll standards, controls, and samples were added and incubated for 2 h. After washing with buffer, 100 ll of standard zero were added to each well, 50 ll HRP-conjugate were added and incubated for 2 h. After washing with buffer, 200 ll TMB chromogenic solutions were added to each well and incubated for 30 min. Fifty microliters of stop solution were added to each well and the absorbance was measured at 450 nm. 2.5. Measurement of serum IgE by ELISA Serum IgE was measured by ELISA kit (Elitech diagnostics – France). 3. Results This study is a prospective study that included 29 patients (19 patients with allergic rhinitis and 10 patients as control). The patients were classified into three groups. Group I (10 patients) with severe allergic rhinitis, group II (9 patients) with mild allergic rhinitis and group III control group (10 patients). They were 13 males and 17 females with mean age 29 ± 7 years. From this study, the level of the eotaxin was higher in severe allergic rhinitis (33.6 pg/ml) than in mild allergic rhinitis (9.8 pg/ml) or control group (0.6 pg/ml) (Table 1). Also, the level of the RANTES and TNF-1 were higher in severe allergic rhinitis (72.17 and 25.47 pg/ml) than in mild allergic rhinitis (10.5 and 12.99 pg/ml) or control group (0.65 and 0.63 pg/ml). Total level of IgE was higher in severe allergic rhinitis (626 IU/ml) than in mild allergic rhinitis (593.9 IU/ml) or control group (31.5 IU/ml). The mean values of eotaxin,
RANTES, TNF-1 and IgE in severe allergic rhinitis are statistically higher than in mild allergic rhinitis and the mean values of all these parameters were higher in both groups compared to control group. By studying the correlation between eotaxin, RANTES and TNF-1 in severe allergic rhinitis, there was no positive correlation between these cytokines in severe allergic rhinitis (Table 2). In mild allergic rhinitis, there was no positive correlation between eotaxin, and RANTES, while positive correlation was found between TNF-1 and both RANTES and eotaxin (Table 3). 4. Discussion Collins et al., 1996, reported that eotaxin had an important local role in eosinophil recruitment from blood microvessels, whereas IL-5 facilitates this process by acting remotely as a hormone to stimulate the release of a rapidly mobilizable pool of bone marrow eosinophils into the circulation.9 Hanazawa et al., 2000, observed that intranasal administration of eotaxin actually increases nasal eosinophils and nitric oxide in patients with allergic rhinitis.10 In our study, the levels of eotaxin, RANTES, and TNF-1 in nasal wash were increased in mild and severe allergic rhinitis compared to normal control, and these cytokines were increased in severe cases compared to mild cases. The increased eotaxin, RANTES, TNF-1 in severe cases suggests another relation of these cytokines to disease severity and suggests a possible role for these cytokines in pathogenesis of allergic rhinitis at the nasal mucosa.
Table 2 Correlations among various parameters in group I (severe allergic rhinitis). Eotaxin
RANTES
TNF-1
1.000 –
0.287 0.421
0.118 0.745
RANTES (pg/ml) Pearson correlation Sig. (2-tailed)
0.287 0.421
1.000 –
0.525 0.119
TNF-1 (pg/ml) Pearson correlation Sig. (2-tailed)
0.118 0.745
0.525 0.119
1.000 –
Eotaxin (pg/ml) Pearson correlation Sig. (2-tailed)
36
A.E. Sharkawy et al.
Table 3 Correlations among various parameters in group II (mild allergic rhinitis). Eotaxin
RANTES
TNF-1
Eotaxin (pg/ml) Pearson correlation Sig. (2-tailed)
1.000 –
0.505 0.137
0.773** 0.009
RANTES (pg/ml) Pearson correlation Sig. (2-tailed)
0.505 0.137
1.000 –
0.885** 0.001
TNF-1 (pg/ml) Pearson correlation Sig. (2-tailed)
0.773** 0.009
0.885** 0.001
1.000 –
**
Correlation is significant at the 0.01 level (2-tailed).
Terada et al., 2001, investigated the levels of eosinophil chemoattractants in nasal lavage fluids obtained after antigen challenge, compared with eosinophil counts and eosinophil protein X (EPX) levels.8 In subjects with allergic rhinitis, allergen challenge led to parallel increases in eosinophil counts, levels of EPX, and eotaxin concentrations in nasal lavage fluid. The levels of eotaxin in lavage samples showed strong correlation with lavage levels of eosinophil counts and EPX. These results indicate that eotaxin has an important role in eosinophil dependent inflammation in nasal mucosa and suggest that blocking eotaxin or CCR-3 might be useful for new therapeutic tools of allergic rhinitis. Sim et al.13 demonstrated that RANTES were released in nasal fluid after allergen challenge in allergic subjects and the levels correlated with corresponding total symptom scores during late-phase response. However, Terada et al. detected RANTES in only 19 of 70 samples of nasal lavage fluids obtained before and after allergen challenge in allergic subjects.8 They indicated that other samples contained RANTES below the sensitivity of the assay they used. Moreover, no significant correlation was observed between RANTES and eosinophil counts or EPX. They concluded that the relative role of RANTES in eosinophilic inflammation in nasal mucosa remains to be established. Correlations were not found between eotaxin and RANTES in severe and mild allergic rhinitis, however, the significant elevations in all these cytokines in the previous conditions indicate that they have complementary functions. Ponath et al.9 indicated that the higher levels of eotaxin immunoreactivity in nasal epithelial cells at sites of active allergic inflammation where cytokines including TNF-1 and IL-1b are known to be available as opposed to adjacent noninflamed, non-cytokine-expressing epithelium imply the existence of a mechanism modulating eotaxin production in allergic airway disease that may involve these cytokines. Bartels et al.14 found elevated expression of eotaxin- and RANTES-mRNA but no MCP-3 mRNA in non-atopic and atopic nasal polyps when compared to normal nasal mucosa. In nasal polyposis, allergic rhinitis and both allergic and nonallergic sinusitis, upregulation of eotaxin mRNA and protein has been detected.6 Nonaka et al.15 concluded that a co-stimulus like lipopolysaccharide is necessary for IL-4 to make a strong induction of eotaxin in eosinophilic inflammation.
Recent studies indicate that (YM-355179) is a novel, selective, and orally available CCR3 antagonist (chemokine receptor antagonist) with therapeutic potential for treating eosinophil-related allergic inflammatory diseases.16 Zhang et al.17 stated that the T helper cell (T(H)) immune system is not well regulated in allergic rhinitis. Most of the upregulated genes are of chemokines and their receptors that play important roles in T(H)2 response, and some are involved in the induction of allergic reaction, accumulation of inflammatory cells, and degranulation of sensitized cells. These findings can point to new strategies for allergic rhinitis immunotherapy. Studies will lead to the identification of novel CCR3 chemokine receptor antagonists, which can be therapeutically used in allergic asthma, allergic rhinitis, and atopic dermatitis.18 In conclusion, we suggest possible roles for eotaxin, RANTES and TNF-1 at the local level in the pathogenesis of allergic rhinitis and their possible relation to disease severity which direct the attention to therapeutic trials for these locally produced cytokines. References 1. Griffiths-Johnson DA, Collins PD, Jose PJ, et al. Animal models of asthma: role of chemokines. Methods Enzymol. 1997;288:241–266. 2. Brown JR, Kleimberg J, Marini M, et al.. Kinetics of eotaxin expression and its relationship to eosinophil accumulation and activation in bronchial biopsies and bronchoalveolar lavage of asthmatic patients after allergen inhalation. Clin Exp Immunol. 1998;114:137–146. 3. Baggiolini M. Eotaxin: a VIC (very important chemokine) of allergic inflammation? J Clin Invest. 1996;97:587. 4. Yao T, Kojima Y, Koyanagi A, et al. Eotaxin-1, -2, and -3 immunoreactivity and protein concentration in the nasal polyps of eosinophilic chronic rhinosinusitis patients. Laryngoscope. 2009;119(6):1053–1059. 5. Olze H, Fo¨rster U, Zuberbier T, Morawietz L, Luger EO. Eosinophilic nasal polyps are a rich source of eotaxin, eotaxin-2 and eotaxin-3. Rhinology. 2006;44(2):145–150. 6. Rojas-Ramos E, Avalos AF, Perez-Fernandez L, et al. Role of the chemokines RANTES, monocyte chemotactic proteins-3 and -4, and eotaxins-1 and -2 in childhood asthma. Eur Respir J. 2003;22: 310–316. 7. Fujikura T, Shimosawa T, Yakuo I. Regulatory effect of histamine H1 receptor antagonist on the expression of messenger RNA encoding CC chemokines in the human nasal mucosa. J Allergy Clin Immunol. 2001;107(1):123–128. 8. Semik-Orzech A, Barczyk A, Wiaderkiewicz R. Interleukin 17, RANTES levels in induced sputum of patients with allergic rhinitis after a single nasal allergen challenge. Ann Allergy Asthma Immunol. 2009;103(5):418–424. 9. Ponath PD, Qin S, Ringler DJ, et al. Cloning of the human eosinophil chemoattractant, eotaxin: expression, receptor binding, and functional properties suggest a mechanism for the selective recruitment of eosinophils. J Clin Invest. 1996;97:604–612. 10. Lilly CM, Nakamura H, Kesselman H, et al. Expression of eotaxin by human lung epithelial cells. Induction by cytokines and inhibition by glucocorticoids. J Clin Invest. 1997;99(7):1767–1773. 11. Widegren H, Erjefa¨lt J, Korsgren M, et al. Effects of intranasal TNFalpha on granulocyte recruitment and activity in healthy subjects and patients with allergic rhinitis. Respir Res. 2008;30: 9–15. 12. Lund VJ, Mackay IS. Staging in rhinosinusitis. Rhinology. 1993;31:183–184.
Eotaxin, RANTES and tumor necrosis factor alpha levels in allergic rhinitis 13. Sim TC, Reece LM, Hilameier KA, et al. Secretion of chemikines and other cytokines in allergen-induced nasal responses: inhibition by topical steroid treatment. Am J respire Crit care Med. 1995;52:927–933. 14. Bartels J, Maune S, Meyer JE, et al. Increased eotaxin-mRNA expression in non-atopic and atopic nasal polyps: comparison to RANTES and MCP-3 expression. Rhinology. 1997;35(4):171–174. 15. Nonaka M, Pawankar R, Fukumoto A, et al. Induction of eotaxin production by interleukin-4, interleukin-13 and lipopolysaccharide by nasal fibroblasts. Clin Exp Allergy. 2004;34:804–811.
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16. Morokata T, Suzuki K, Masunaga Y. Novel, selective, and orally available antagonist for CC chemokine receptor 3. J Pharmacol Exp Ther. 2006;317:244–250, Epub 2005 Dec 9. 17. Zhang RX, Yu SQ, Jiang JZ, Liu GJ. Complementary DNA microarray analysis of chemokines and their receptors in allergic rhinitis. J Investig Allergol Clin Immunol. 2007;17(5):329–336. 18. Elsner J, Escher SE, Forssmann U. Chemokine receptor antagonists: a novel therapeutic approach in allergic diseases. Allergy. 2004;59(12):1243–1258.
Egyptian Society of Ear, Nose, Throat and Allied Sciences
Egyptian Journal of Ear, Nose, Throat and Allied Sciences www.esentas.org
ORIGINAL ARTICLE
Eotaxin, RANTES and tumor necrosis factor alpha levels in allergic rhinitis Asser El Sharkawy a b
a,*
, Shawky Elmorsy a, Mohammed El-Naggar
b
Department of Otorhinolaryngology, Faculty of Medicine, Mansoura University, Mansoura, Egypt Department of Medical Microbiology & Immunology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
Received 11 December 2010; accepted 9 February 2011 Available online 23 June 2011
KEYWORDS Allergic rhinitis; Chemokines; Tumor necrosis factor alpha
Abstract Objectives: The objectives of this study were to estimate the levels of eotaxin, RANTES and tumor necrosing factor-1 in allergic rhinitis and their relation to disease severity. Study design: Prospective study. Setting: Mansoura University Hospital. Patients and methods: Twenty nine patients suffering from allergic rhinitis were included in this study (19 patients with allergic rhinitis and 10 patients control group). The patients underwent estimation of eotaxin, RANTES, TNF-1 in the nasal wash using Elisa technique. The patients were divided according to the disease severity into mild allergic rhinitis, severe allergic rhinitis and control groups. Results: The mean values of eotaxin, RANTES, TNF-1 in severe allergic rhinitis (33.6 ± 11.07 pg/ml, 72.17 ± 87.61 pg/ml, 25.47 ± 4.04 pg/ml) were statistically higher than in mild allergic rhinitis (9.80 ± 6.79 pg/ml, 10.50 ± 6.90 pg/ml, 12.99 ± 3.27 pg/ml) and the mean values of all these parameters were higher in both groups compared to control group (0.6 ± 0.69 pg/ml, 0.65 ± 0.74 pg/ml, 0.63 ± 0.54 pg/ml).
* Corresponding author. Address: ORL Department, Mansoura University Hospital, Mansoura, Egypt. Tel.: +20 102223269; fax: +20 502267016. E-mail address: [email protected] (A.El Sharkawy). 2090-0740 ª 2011 Egyptian Society of Ear, Nose, Throat and Allied Sciences. Production and hosting by Elsevier B.V. All rights reserved. Peer review under responsibility of Egyptian Society of Ear, Nose, Throat and Allied Sciences. doi:10.1016/j.ejenta.2011.02.001
Production and hosting by Elsevier
34
A.E. Sharkawy et al. Conclusion: This study suggests the role of local chemokines in the pathogenesis of allergic rhinitis as well as their possible relation to the severity of the disease which may direct the attention to therapeutic trials against these locally produced chemokines. ª 2011 Egyptian Society of Ear, Nose, Throat and Allied Sciences. Production and hosting by Elsevier B.V. All rights reserved.
1. Introduction
2. Patients and methods
There is accumulating evidence that chemokines are involved in both the migration and the activation of eosinophil and other leukocytes during asthma responses. These chemokines include RANTES, macrophage-inflammatory protein-11 (MIP-11), monocyte-chemotactic protein-3 (MCP-3), eotaxin, and MCP-5.1 Eotaxin is the major eosinophil chemoattractant that binds with high affinity to chemokin receptor 3 (CCR3) which is expressed on cells such as eosinophils, basophils, and Th-2 lymphocytes.2 It is secreted by multiple cell types including epithelium, fibroblasts, smooth muscle cells, and eosinophils.3 Eotaxin family production by eosinophils results in the recruitment of eosinophils into the tissue by a self-amplifying process in eosinophilic chronic rhinosinusitis patients.4 All members of the Eotaxin family (Eotaxin-1, -2 and -3) are involved in the pathogenesis of nasal polyposis. The results are more likely indicative of a complex cooperation between all members of the geotaxis family than of a specific role in the development of eosinophilia and nasal polyposis.5 RANTES is a broad-acting chemokine that has an established role in the recruitment of CD4+ T cells, monocytes, and eosinophils into inflammatory sites, and is upregulated in patients with asthma. It is produced by numerous lung cell types including CD8+ T cells and bronchial epithelial cells.6 Fujikura et al.7 investigated the expression of RANTES, and eotaxin in the nasal mucosa of patients with allergic rhinitis to clarify the effect of histamine and antihistamine on the regulation of the expression of these Cysteine–Cysteine (CC) chemokines. They concluded that histamine may induce CC chemokine production in the nasal mucosa of patients with allergic rhinitis. They suggested that the regulation of histamine-CC chemokine interaction could lead to new therapeutic approaches for the treatment of nasal allergy. Nasal allergen challenge induces the enhanced secretion of IL-17 and RANTES in the lower airways of nonasthmatic patients with allergic rhinitis.8 Although TNF-1 can cause the characteristic physiological phenotype of asthma, namely airway hyper responsiveness and eosinophilia, the fact that it is not a potent chemoattractant for eosinophils suggests that its effects on eosinophil recruitment are indirect.9 Lilly et al.10 demonstrates that TNF-1 and IL-1b rapidly and transiently induce the accumulation of eotaxin mRNA in bronchial epithelial cell lines and that this increase is associated with the intracellular accumulation of eotaxin protein and its secretion into the cell supernatant. TNF-1 produces a nasal inflammatory response in humans that is characterized by late phase (i.e., 24 h post challenge) neutrophil activity and plasma exudation.11 The aim of this study was to evaluate the levels of eotaxin, RANTES and tumor necrosing factor-1 in allergic rhinitis and their possible relation to disease severity.
Ethical approval for this work was obtained by the Ethical Committee Board of Faculty of Medicine, Mansoura University. This work was done in ORL department and Microbiology and immunology departments in the period between August 2007 and May 2008. The study included 29 patients (19 patients with allergic rhinitis and 10 control). The patients were selected from the out patients clinic of the ORL department Mansoura university. The diagnosis of allergic rhinitis was confirmed by clinical history, clinical examination, and positive skin test for certain allergen and elevated total serum IgE. The patients were classified into three groups: group I (n = 10) patients with severe allergic symptoms, group II (n = 9) patients with mild allergic symptoms based on visual analogue scale (VAS).12 The previous groups were compared with nonsmoking subjects with no positive skin-prick response to common aeroallergens or history of respiratory disease acted as a control group III (n = 10). 2.1. Nasal wash samples The nasal cavity was washed with 10 ml of physiological saline warmed at 37 C by reciprocating the piston of the syringe 10 times on each side. Lavage fluid was centrifuged for 20 min at 400·g. The supernatant was stored frozen at 30 C until assayed.8 2.2. Measurement of eotaxin Eotaxin was measured in nasal wash using ELISA technique. The BioSource human Eotaxin (hEotaxin) ELISA was used (BioSource, Nivelles, Belgium). Fifty microliters of the standard diluent buffer were added to the wells reserved for samples and zero well. Fifty microliters of standards, samples or controls were added to the appropriate microtiter wells. One hundred microliters of biotinylated anti-eotaxin solution were pipette into each well. After incubation for 2 h, wells were aspirated and washed. One hundred microliters Streptavidin-HRP working solution were added to each well and incubated for 30 min and thereafter, wells were thoroughly aspirated and washed. One hundred microliters of stabilized chromogen were added to each well. After incubation for 30 min, 100 ll of stop solution were added to each well. Absorbance of each well was read at 450 nm. 2.3. Measurement of RANTES RANTES was measured in nasal wash and polyp samples using ELISA technique. The BioSource human RANTES (hRANTES) ELISA was used (BioSource, Nivelles, Belgium). Fifty microliters of the standard diluent buffer were added to zero wells. Fifty microliters of standards, samples or controls
Eotaxin, RANTES and tumor necrosis factor alpha levels in allergic rhinitis Table 1
35
Eotaxin, Rantes and TNF-1 and total serum IgE in different groups.
Group I Group II Group III (control group) P* P** P***
Eotaxin (pg/ml)
RANTES (pg/ml)
TNF-1 (pg/ml)
Total IgE (IU/ml)
M
M
M
SD
M
SD
4.04 3.27 0.54
626 593.9 31.5 0.803 <0.001 <0.001
281.78 286.23 11.03
33.6 9.80 0.6 <0.001 <0.001 <0.001
SD 11.07 6.79 0.69
72.17 10.50 0.65 0.04 0.019 <0.001
SD 87.61 6.90 0.74
25.47 12.99 0.63 <0.001 <0.001 <0.001
*
P (severe allergic rhinitis vs. mild allergic rhinitis). P (severe allergic rhinitis vs. control). *** P (mild allergic rhinitis vs. control). **
were added to the appropriate microtiter wells and 50 ll of biotinylated anti-RANTES solution were pipette into each well and incubated for 1 h. Wells were aspirated and washed. One hundred microliters Streptavidin-HRP working solution was added to each well. After incubation for 30 min, wells were aspirated and washed and 100 ll of stabilized chromogen were added to each well. After incubation for 30 min, 100 ll of stop solution were added to each well. Absorbance of each well was read at 450 nm. 2.4. Measurement of tumor necrosis factor-1 EASIA TM ELISA Kit (Biosource, Nivelles, Belgium) was used. Fifty microliters incubation buffers were added to all wells, 200 ll standards, controls, and samples were added and incubated for 2 h. After washing with buffer, 100 ll of standard zero were added to each well, 50 ll HRP-conjugate were added and incubated for 2 h. After washing with buffer, 200 ll TMB chromogenic solutions were added to each well and incubated for 30 min. Fifty microliters of stop solution were added to each well and the absorbance was measured at 450 nm. 2.5. Measurement of serum IgE by ELISA Serum IgE was measured by ELISA kit (Elitech diagnostics – France). 3. Results This study is a prospective study that included 29 patients (19 patients with allergic rhinitis and 10 patients as control). The patients were classified into three groups. Group I (10 patients) with severe allergic rhinitis, group II (9 patients) with mild allergic rhinitis and group III control group (10 patients). They were 13 males and 17 females with mean age 29 ± 7 years. From this study, the level of the eotaxin was higher in severe allergic rhinitis (33.6 pg/ml) than in mild allergic rhinitis (9.8 pg/ml) or control group (0.6 pg/ml) (Table 1). Also, the level of the RANTES and TNF-1 were higher in severe allergic rhinitis (72.17 and 25.47 pg/ml) than in mild allergic rhinitis (10.5 and 12.99 pg/ml) or control group (0.65 and 0.63 pg/ml). Total level of IgE was higher in severe allergic rhinitis (626 IU/ml) than in mild allergic rhinitis (593.9 IU/ml) or control group (31.5 IU/ml). The mean values of eotaxin,
RANTES, TNF-1 and IgE in severe allergic rhinitis are statistically higher than in mild allergic rhinitis and the mean values of all these parameters were higher in both groups compared to control group. By studying the correlation between eotaxin, RANTES and TNF-1 in severe allergic rhinitis, there was no positive correlation between these cytokines in severe allergic rhinitis (Table 2). In mild allergic rhinitis, there was no positive correlation between eotaxin, and RANTES, while positive correlation was found between TNF-1 and both RANTES and eotaxin (Table 3). 4. Discussion Collins et al., 1996, reported that eotaxin had an important local role in eosinophil recruitment from blood microvessels, whereas IL-5 facilitates this process by acting remotely as a hormone to stimulate the release of a rapidly mobilizable pool of bone marrow eosinophils into the circulation.9 Hanazawa et al., 2000, observed that intranasal administration of eotaxin actually increases nasal eosinophils and nitric oxide in patients with allergic rhinitis.10 In our study, the levels of eotaxin, RANTES, and TNF-1 in nasal wash were increased in mild and severe allergic rhinitis compared to normal control, and these cytokines were increased in severe cases compared to mild cases. The increased eotaxin, RANTES, TNF-1 in severe cases suggests another relation of these cytokines to disease severity and suggests a possible role for these cytokines in pathogenesis of allergic rhinitis at the nasal mucosa.
Table 2 Correlations among various parameters in group I (severe allergic rhinitis). Eotaxin
RANTES
TNF-1
1.000 –
0.287 0.421
0.118 0.745
RANTES (pg/ml) Pearson correlation Sig. (2-tailed)
0.287 0.421
1.000 –
0.525 0.119
TNF-1 (pg/ml) Pearson correlation Sig. (2-tailed)
0.118 0.745
0.525 0.119
1.000 –
Eotaxin (pg/ml) Pearson correlation Sig. (2-tailed)
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A.E. Sharkawy et al.
Table 3 Correlations among various parameters in group II (mild allergic rhinitis). Eotaxin
RANTES
TNF-1
Eotaxin (pg/ml) Pearson correlation Sig. (2-tailed)
1.000 –
0.505 0.137
0.773** 0.009
RANTES (pg/ml) Pearson correlation Sig. (2-tailed)
0.505 0.137
1.000 –
0.885** 0.001
TNF-1 (pg/ml) Pearson correlation Sig. (2-tailed)
0.773** 0.009
0.885** 0.001
1.000 –
**
Correlation is significant at the 0.01 level (2-tailed).
Terada et al., 2001, investigated the levels of eosinophil chemoattractants in nasal lavage fluids obtained after antigen challenge, compared with eosinophil counts and eosinophil protein X (EPX) levels.8 In subjects with allergic rhinitis, allergen challenge led to parallel increases in eosinophil counts, levels of EPX, and eotaxin concentrations in nasal lavage fluid. The levels of eotaxin in lavage samples showed strong correlation with lavage levels of eosinophil counts and EPX. These results indicate that eotaxin has an important role in eosinophil dependent inflammation in nasal mucosa and suggest that blocking eotaxin or CCR-3 might be useful for new therapeutic tools of allergic rhinitis. Sim et al.13 demonstrated that RANTES were released in nasal fluid after allergen challenge in allergic subjects and the levels correlated with corresponding total symptom scores during late-phase response. However, Terada et al. detected RANTES in only 19 of 70 samples of nasal lavage fluids obtained before and after allergen challenge in allergic subjects.8 They indicated that other samples contained RANTES below the sensitivity of the assay they used. Moreover, no significant correlation was observed between RANTES and eosinophil counts or EPX. They concluded that the relative role of RANTES in eosinophilic inflammation in nasal mucosa remains to be established. Correlations were not found between eotaxin and RANTES in severe and mild allergic rhinitis, however, the significant elevations in all these cytokines in the previous conditions indicate that they have complementary functions. Ponath et al.9 indicated that the higher levels of eotaxin immunoreactivity in nasal epithelial cells at sites of active allergic inflammation where cytokines including TNF-1 and IL-1b are known to be available as opposed to adjacent noninflamed, non-cytokine-expressing epithelium imply the existence of a mechanism modulating eotaxin production in allergic airway disease that may involve these cytokines. Bartels et al.14 found elevated expression of eotaxin- and RANTES-mRNA but no MCP-3 mRNA in non-atopic and atopic nasal polyps when compared to normal nasal mucosa. In nasal polyposis, allergic rhinitis and both allergic and nonallergic sinusitis, upregulation of eotaxin mRNA and protein has been detected.6 Nonaka et al.15 concluded that a co-stimulus like lipopolysaccharide is necessary for IL-4 to make a strong induction of eotaxin in eosinophilic inflammation.
Recent studies indicate that (YM-355179) is a novel, selective, and orally available CCR3 antagonist (chemokine receptor antagonist) with therapeutic potential for treating eosinophil-related allergic inflammatory diseases.16 Zhang et al.17 stated that the T helper cell (T(H)) immune system is not well regulated in allergic rhinitis. Most of the upregulated genes are of chemokines and their receptors that play important roles in T(H)2 response, and some are involved in the induction of allergic reaction, accumulation of inflammatory cells, and degranulation of sensitized cells. These findings can point to new strategies for allergic rhinitis immunotherapy. Studies will lead to the identification of novel CCR3 chemokine receptor antagonists, which can be therapeutically used in allergic asthma, allergic rhinitis, and atopic dermatitis.18 In conclusion, we suggest possible roles for eotaxin, RANTES and TNF-1 at the local level in the pathogenesis of allergic rhinitis and their possible relation to disease severity which direct the attention to therapeutic trials for these locally produced cytokines. References 1. Griffiths-Johnson DA, Collins PD, Jose PJ, et al. Animal models of asthma: role of chemokines. Methods Enzymol. 1997;288:241–266. 2. Brown JR, Kleimberg J, Marini M, et al.. Kinetics of eotaxin expression and its relationship to eosinophil accumulation and activation in bronchial biopsies and bronchoalveolar lavage of asthmatic patients after allergen inhalation. Clin Exp Immunol. 1998;114:137–146. 3. Baggiolini M. Eotaxin: a VIC (very important chemokine) of allergic inflammation? J Clin Invest. 1996;97:587. 4. Yao T, Kojima Y, Koyanagi A, et al. Eotaxin-1, -2, and -3 immunoreactivity and protein concentration in the nasal polyps of eosinophilic chronic rhinosinusitis patients. Laryngoscope. 2009;119(6):1053–1059. 5. Olze H, Fo¨rster U, Zuberbier T, Morawietz L, Luger EO. Eosinophilic nasal polyps are a rich source of eotaxin, eotaxin-2 and eotaxin-3. Rhinology. 2006;44(2):145–150. 6. Rojas-Ramos E, Avalos AF, Perez-Fernandez L, et al. Role of the chemokines RANTES, monocyte chemotactic proteins-3 and -4, and eotaxins-1 and -2 in childhood asthma. Eur Respir J. 2003;22: 310–316. 7. Fujikura T, Shimosawa T, Yakuo I. Regulatory effect of histamine H1 receptor antagonist on the expression of messenger RNA encoding CC chemokines in the human nasal mucosa. J Allergy Clin Immunol. 2001;107(1):123–128. 8. Semik-Orzech A, Barczyk A, Wiaderkiewicz R. Interleukin 17, RANTES levels in induced sputum of patients with allergic rhinitis after a single nasal allergen challenge. Ann Allergy Asthma Immunol. 2009;103(5):418–424. 9. Ponath PD, Qin S, Ringler DJ, et al. Cloning of the human eosinophil chemoattractant, eotaxin: expression, receptor binding, and functional properties suggest a mechanism for the selective recruitment of eosinophils. J Clin Invest. 1996;97:604–612. 10. Lilly CM, Nakamura H, Kesselman H, et al. Expression of eotaxin by human lung epithelial cells. Induction by cytokines and inhibition by glucocorticoids. J Clin Invest. 1997;99(7):1767–1773. 11. Widegren H, Erjefa¨lt J, Korsgren M, et al. Effects of intranasal TNFalpha on granulocyte recruitment and activity in healthy subjects and patients with allergic rhinitis. Respir Res. 2008;30: 9–15. 12. Lund VJ, Mackay IS. Staging in rhinosinusitis. Rhinology. 1993;31:183–184.
Eotaxin, RANTES and tumor necrosis factor alpha levels in allergic rhinitis 13. Sim TC, Reece LM, Hilameier KA, et al. Secretion of chemikines and other cytokines in allergen-induced nasal responses: inhibition by topical steroid treatment. Am J respire Crit care Med. 1995;52:927–933. 14. Bartels J, Maune S, Meyer JE, et al. Increased eotaxin-mRNA expression in non-atopic and atopic nasal polyps: comparison to RANTES and MCP-3 expression. Rhinology. 1997;35(4):171–174. 15. Nonaka M, Pawankar R, Fukumoto A, et al. Induction of eotaxin production by interleukin-4, interleukin-13 and lipopolysaccharide by nasal fibroblasts. Clin Exp Allergy. 2004;34:804–811.
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