ARTICLE INFO

Article Type

Original Research

Authors

Ghaffarpour   S. (1 )
Ghazanfari   T. (* )
Salehi   I. (2 )
Askary   N. (3 )
Mir Afshariyeh   A. (4 )
Faghihzadeh   E. (1 )






(* ) Immunoregulation Research Center, Shahed University, Tehran, Iran
(1 ) Immunoregulation Research Center, Shahed University, Tehran, Iran
(2 ) Immunology Department, Medicine Faculty, Tehran University, Tehran, Iran
(3 ) Biology Department, Sciences Faculty, Shahid Bahonar University of Kerman, Kerman, Iran
(4 ) Pathology Department, Medicine Faculty, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Correspondence

Article History

Received:  July  9, 2014
Accepted:  August 25, 2014
ePublished:  September 20, 2014

BRIEF TEXT


Acute (short-term) and chronic (long-term) complications can occur after mustard gas exposure. According to various studies, the most common delayed complications are pulmonary disorders, including chronic obstructive pulmonary disease (COPD), chronic bronchitis (CB), bronchiolitis obliterans (BO), bronchiectasis, and increase in pulmonary response [1]. There are few studies regarding lung pathology of chemically-injured persons. However, according to these studies, it seems that bronchiolitis obliterans is the main consequence of mustard gas exposure [2]. Having specific characteristics, respiratory disorders in chemically-injured persons are known under the title “mustard lung” [3-5]. According to various studies, there are some changes in the major factors involved in inflammation in serum and sputum samples of chemically-injured persons, 20 years after mustard gas exposure [6-12]. … [13] Some researchers have reported diversity of bacteria types, which were mostly of respiratory tract flora, in patients with delayed complications of mustard gas exposure [14]. In addition, there is a significant difference in IL-8, IL-1β, IL-6, TNFα, and IL-12 inflammatory levels in BAL between bronchiectasis patients with mustard gas exposure and healthy persons [15]. … [16-18]

Change in some gene expressions in rats with mustard gas exposure has been shown [19], also, there are some researches about pulmonary complications in chemically-injured peoples exposed to mustard gas in Iran, showing increase in MMP-9 among the patients than healthy peoples, which has a reverse relation with FVC [8, 20]. Nevertheless, there is no study on TLR4 assessment in lung samples of chemically-injured veterans, despite the fact that there are various reports about change in expression of the indicator in chronic pulmonary inflammatory diseases [21-23].

The aim of this study was to evaluate the level of TLR4 gene expression in lung’s tissue of chemically-injured veterans with delayed pulmonary complications due to mustard gas.

This is a case-control study.

Paraffin blocks of lung tissue samples from 28 veterans exposed to mustard gas with delayed pulmonary complications (case group) and 9 pulmonary samples from the subjects with no mustard exposure (control group) were collected from the archives of Hazrat-e-Rasool and Mahdieh hospitals in 2013-2014. The used blocks were prepared during 5 to 10 years leading to the study, in order to diagnose the disease.

Inclusion criteria were male gender, age between 30 to 60 years, and exposed to mustard gas during Iran-Iraq war according to confirmed documents of Martyrs and Veterans Foundation’s Medical Commission with diagnosis of common delayed pulmonary complications like cough, disposal of humors, and dyspnea due to respiratory distress, registered in medical records.

Slide samples from lung’s paraffin blocks, were stained with Hematoxyline and Eosin (H&E) [18]; and after histopathological diagnosis by a pulmonary pathologist, they were divided into ‘bronchiolitis obliterans’ and ‘respiratory bronchiolitis’ groups. Real-time PCR method was used to evaluate RNA expression. The samples’ concentration and absorption were measured with spectrophotometer 2000c UV-Vis Nano drop (Thermo Scientific; USA). From each RNA sample, 1 microgram was transferred into cDNA with PrimeScript RT reagent kit (Takara Bio Inc; Japan). Then, TLR4 and GAPDH (as the reference gene) expressions were assessed through primers with confirmed specificity in BLAST (Table 1), and using SYBR® Premix Ex Taq II master mix (Takara Bio Inc; Japan) and with StepOnePlus instrument (ABI System; USA). ∆∆CT Method was used to assess relatively the genes’ expressions [24].‏ Through cDNA Sample dilution, return of the two genes was performed by 1:5 and their difference reached less than 5%. Mann-Whitney Test and SPSS 21 were used to compare data.

At sampling time, average ages of ‘case’ and ‘control’ groups were41.95±11.13 and 49.72±11.64 years, respectively. From ‘case’ and ‘control’ groups in this arrangement, three pairs including 12 (42.8%) and 4 (44.4%) patients, 8 (28.6%) and 1 (11.2%) patients, and 8 (28.6%) and 4 (44.4%) patients were diagnosed with bronchiolitis obliterans, respiratory bronchiolitis, and other diseases, respectively. A_260 by A_280 absorption ratio of the samples was 1.86±0.12. In ‘case’ and ‘control’ groups, ∆∆CT Averages, without any significant difference, were 5.28±3.58cycle and5.81±3.29cycle respectively. Using 2^(-∆∆CT)formula, TLR4 expression reduction was observed in ‘case’ group by 1:0.49, than ‘control’ group. This difference was not significant.

Results of the present study are consistent with results of some other studies, reporting bronchiolitis obliterans as the main delayed pulmonary complications in patients exposed to mustard gas [2]. After one time exposure of rat skin to mustard gas, increase in TLR4 expression after 72 hours and 168 hours (delayed phase) was reported [19]. This is not enough time to assess the delayed phase. In addition, the used tissue in mustard gas exposure was different with the tissue used in the present study. … [25-28]. Increase in TLR4 in peripheral and central airways of the smoker patients, peoples who quit smoking, and control smoker subjects than control healthy subjects with no smoking history via immunohistochemical method has been reported. In addition, increase in expression of β-defensins, produced after TLR4 stimulation, has been shown in the peripheral airway. Notice that all the samples have been gathered from patients with pulmonary cancer [29]. In another study, increase in TLR4 and TLR9 expression in CD8+T cells in the lung samples of patients with COPD than healthy persons was reported. However, notice that there has been no significant change in TLR4 expression in the total tissue [30].

It is suggested that this study to be continued with more samples and with comparative evaluation with healthy samples.

Few chemically-injured persons and unavailability of healthy control samples were of limitations of this study.

TLR4 gene expression in lung tissues of chemically-injured persons and other patients with pulmonary diseases is similar to the pathology of bronchiolitis obliterans.

Non-declared

Non-declared

Non-declared

Non-declared

TABLES and CHARTS

Show attach file


CITIATION LINKS

[1]Lari SM, Attaran D, Towhidi M. COPD Due to Sulfur Mustard (Mustard Lung). In: Ong KCh, editor. Chronic Obstructive Pulmonary Disease - Current Concepts and Practice. Croatia: InTech; 2012. pp. 231-8.
[2]Ghanei M, Chilosi M, Hosseini Akbari HM, Motiei-Langroudi R, Amini Harandi A, Shamsaei H, et al. Use of immunohistochemistry techniques in patients exposed to sulphur mustard gas. Pathol Res Int. 2011. Available From: http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3138111.
[3]Ghanei M, Harandi AA. Molecular and cellular mechanism of lung injuries due to exposure to sulfur mustard: A review. Inhal Toxicol. 2011;23(7):363-71.
[4]Razavi SM, Ghanei M, Salamati P, Safiabadi M. Long-term effects of mustard gas on respiratory system of Iranian veterans after Iraq-Iran war: A review. Chinese J Traumatol. 2013;16(3):163-8.
[5]Razavi SM, Ghanei M, Salamati P, Safiabadi M. Long-term effects of mustard gas on respiratory system of Iranian veterans after Iraq-Iran war: A review. Chinese J Traumatol. 2013;16(3):163-8.
[6]Rowell M, Kehe K, Balszuweit F, Thiermann H. The chronic effects of sulfur mustard exposure. Toxicology. 2009;263(1):9-11.
[7]Ghazanfari T, Yaraee R, Kariminia A, Ebtekar M, Faghihzadeh S, Vaez-Mahdavi MR, et al. Alterations in the serum levels of chemokines 20 years after sulfur mustard exposure: Sardasht-Iran Cohort Study. Int Immunopharmacol. 2009;9(13-14):1471-6.
[8]Pourfarzam S, Ghazanfari T, Yaraee R, Ghasemi H, Hassan ZM, Faghihzadeh S, et al. Serum levels of IL-8 and IL-6 in the long term pulmonary complications induced by sulfur mustard: Sardasht-Iran Cohort Study. Int Immunopharmacol. 2009;9(13-14):1482-8.
[9]Pourfarzam S, Yaraee R, Hassan ZM, Yarmohammadi ME, Faghihzadeh S, Soroush MR, et al. Chemokines, MMP-9 and PMN elastase in spontaneous sputum of sulfur mustard exposed civilians: Sardasht-Iran Cohort Study. Int Immunopharmacol. 2013;17(3):958-63.
[10]Yaraee R, Ghazanfari T, Ebtekar M, Ardestani SK, Rezaei A, Kariminia A, et al. Alterations in serum levels of inflammatory cytokines (TNF, IL-1alpha, IL-1beta and IL-1Ra) 20 years after sulfur mustard exposure: Sardasht-Iran cohort study. Int Immunopharmacol. 2009;9(13-14):1466-70.
[11]Yaraee R, Ghazanfari T, Faghihzadeh S, Mostafaie A, Soroush MR, Inai K, et al. Alterations in the serum levels of soluble L, P and E-selectin 20 years after sulfur mustard exposure: Sardasht-Iran Cohort Study. Int Immunopharmacol. 2009;9(13-14):1477-81.
[12]Kiani A, Mostafaie A, Shirazi FH, Ghazanfari T. Serum profiles of matrix metalloproteinases and their tissue inhibitors in long-term pulmonary complication induced by sulfur mustard: Sardasht-Iran Cohort Study (SICS). Int Immunopharmacol. 2013;17(3):964-7.
[13]Yaraee R, Hassan ZM, Pourfarzam S, Rezaei A, Faghihzadeh S, Ebtekar M, et al. Fibrinogen and inflammatory cytokines in spontaneous sputum of sulfur-mustard-exposed civilians--Sardasht-Iran Cohort Study. Int Immunopharmacol. 2013;17(3):968-73.
[14]Ghanei M, Adibi I. Clinical Review of Mustard Lung. IJMS. 2007;32(2):58-65.
[15]Panahi Y, Ghanei M, Aslani J, Mojtahedzadeh M, Sarhangnejad R, Barkhordari A. Evaluation of microbial resistance to antibiotics in patients with chronic pulmonary lesions due to chemical agents and non-chemical agents. J Mil Med. 2004;6(3):181-6. [Persian]
[16]Emad A, Emad Y. Levels of cytokine in bronchoalveolar lavage (BAL) fluid in patients with pulmonary fibrosis due to sulfur mustard gas inhalation. J Interferon Cytokine Res. 2007;27(1):38-43.
[17]Joeong E, Lee J. Intrinsic and extrinsic regulation of innate immune receptors. Yonsei Med J. 2011;52(3):379-92.
[18]Takeda K, Kaisho T, Akira Sh. Toll-like receptors. Ann Rev Immunol. 2003;21:335-76.
[19]Plummer LE, Smiley-Jewell S, Pinkerton KE. Impact of air pollution on lung inflammation and the role of Toll-like receptors. Int J Interferon Cytokine Mediator Res. 2012;4:43-57.
[20]Gerecke DR, Chen M, Isukapalli SS, Gordon MK, Chang YC, Tong W, et al. Differential gene expression profiling of mouse skin after sulfur mustard exposure: Extended time response and inhibitor effect. Toxicol Appl Pharmacol. 2009;234(2):156-65.
[21]Ghaffarpour S, Ghazanfari T. Correlation between MMP9 and MMP9/ TIMPs complex with pulmonary function in sulfur mustard exposed civilians: Sardasht-Iran Cohort Study. Iran J Immunol. 2014;11:458-9.
[22]Bezemer GF, Sagar S, van Bergenhenegouwen J, Georgiou NA, Garssen J, Kraneveld AD, et al. Dual role of Toll-like receptors in asthma and chronic obstructive pulmonary disease. Pharmacol Rev. 2012;64(2):337-58.
[23]Go H, Koh J, Kim HS, Jeon YK, Chung DH. Expression of toll-like receptor 2 and 4 is increased in the respiratory epithelial cells of chronic idiopathic interstitial pneumonia patients. Respir Med. 2014;108(5):783-92.
[24]Grossman EJ, Shilling RA. Bronchiolitis obliterans in lung transplantation: The good, the bad, and the future. Transl Res. 2009;153(4):153-65.
[25]Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods. 2001;25(4):402-8.
[26]Chaudhuri N, Dower SK, Whyte MK, Sabroe I. Toll-like receptors and chronic lung disease. Clin Sci. 2005;109(2):125-33.
[27]Ghanei M, Tazelaar HD, Chilosi M, Harandi AA, Peyman M, Akbari HM, Shamsaei H, et al. An international collaborative pathologic study of surgical lung biopsies from mustard gas-exposed patients. Respir Med. 2008;102(6):825-30
[28]Garantziotis S, Palmer S, Snyder L, Ganous T, Chen B, Wang T, et al. Alloimmune lung injury induced by local innate immune activation through inhaled lipopolysaccharide. Transplantation. 2007;84(8):1012-9.
[29]Tesar BM, Jiang D, Liang J, Palmer SM, Noble PW, Goldstein DR. The Role of Hyaluronan Degradation Products as Innate Alloimmune Agonists. Am J Transplant. 2006;6(11):2622-35.
[30]Pace E, Ferraro M, Minervini MI, Vitulo P, Pipitone L, Chiappara G, et al. Beta defensin-2 is reduced in central but not in distal airways of smoker COPD patients. PLoS One. 2012;7(3):e3360.
[31]Nadigel J, Préfontaine D, Baglole CJ, Maltais F, Bourbeau J, Eidelman DH, et al. Cigarette smoke increases TLR4 and TLR9 expression and induces cytokine production from CD8+ T cells in chronic obstructive pulmonary disease. Respir Res. 2011;12(1):149.