About Bioline  All Journals  Testimonials  Membership  News

Medical Journal of The Islamic Republic of Iran
National Research Centre of Medical Sciences of I.R. IRAN
ISSN: 1016-1430
Vol. 20, Num. 2, 2006, pp. 52-56

Medical Journal of the Islamic Republic of Iran , Vol. 20, No. 2, July, 2006, pp. 52-56



From the* Department of Immunology, Faculty of Medicine and Research Center of Molecular Biology, and the ** Research Center of the Chemically Injured, Baqiyatallah University of Medical Sciences, Tehran, I.R. Iran.
* Corresponding author: Department of Immunology, Faculty of Medicine and Research Center of Molecular Biology, Tehran, I.R. IRAN. Email:

Code Number: mr06013


Background: Cytokines play a major role in both acute and chronic inflammatory processes, including those produced by Sulfur Mustard. This study describes the cytokine level six months after exposure to a single dose of sulfur mustard, defined by IL-1β, IL-6, IL-9, IL-12, TGFβ and TNF-α.
Methods: The cytokine levels of Broncho-Alveolar Lavage (BAL) and sera of twenty male rats exposed to sulfur mustard were measured and compared with the control group. The rats in the test group were exposed to a single dose of sulphur mustard (inhalation) and left for up to 6 months. After six months the animals were anesthetised, blood samples were obtained from their heart using 5-mL syringes and serum was kept at -20ºC. Their BAL was collected by lavage. BAL fluid was centrifuged and left at -20ºC until assay. Cytokine assay was performed employing the ELISA Method (Bender Med Systems).
Results: The results showed significant differences (p<0.001) between the control and exposed groups in terms of all cytokine (IL-9, γIFN, TGFβ, IL-6, IL-1β, IL-12 and TNFα) productions in both the BAL fluid and serum. The most noticeable increase in cytokine release was seen in IL-9, which was 615.93% and 321.88% for the BAL fluid and serum, respectively (p<0.001). After IL-9 the highest increase was demonstrated for TGF-β and IL-6 in the BAL fluid which was 200.85% and 125.50% respectively.
Conclusions: From data presented here, it is possible to suggest that over production of IL-6, IL-9 and TGFβ might be involved in the late outcome of lung injury after six months exposure to sulfur mustard.

Keywords: Sulfur Mustard, BAL, Cytokine, Serum, IL-1β, IL-6, IL-9, IL-12, TGFβ and TNF-α.


Based on the cytokine environment present in Interstitial Lung Diseases (ILDs) and the pattern of gene expression of unfractionated Broncho-Alveolar Lavage (BAL) cells from ILD patients with lung inflammation, it appears that the macrophages in lung disease have undergone alternative, rather than classical, activation.1 Recent studies have added to our understanding of the role of cytokine and cytokine receptors in the generation of pulmonary inflammatory responses. In the lung, the production of cytokine and expression of cytokine receptors is under complex biologic control, including negative and positive feedback by the cytokines themselves.2 IL-9 is a multifunctional cytokine produced by activated Th2 cells that promotes inflammation and air-way hyperresponses.3 IFN-γ is a key factor in the events that favour local immune responses in the lung. It activates pulmonary macrophages to phagocytose pathogens. IFN-γ is typically expressed by Th1 cells infiltrating the lung in most ILDs.4 IL-1β is produced by alveolar macrophages in response to several inflammatory stimuli in various ILDs.5 Since IL-1β promotes the proliferation of fibroblast and increased collagen production, it has been involved in the development of lung fibrosis associated with ILDs. In the lung, IL-6 is mostly produced by alveolar macrophages. An increased release of IL-6 has been involved in the pathogenesis of various ILDs.6 IL-12 is mainly produced in the lung by macrophages and dendritic cells. In synergy with IL-15, IL-12 favours the contact between activated T cells and antigen presenting cells (APC).7 TGF-β is a potent immunosuppressive molecule that exerts chemotactic effects on monocytes. It modulates the synthesis and the effect of several molecules, including IL-1, IL-2, IL-3, GM-CSF, IFN-γ and TNF-α. TGF-β, which is constitutively released in the respiratory tract, is involved in the pathogenesis of fibrotic processes associated with most ILDs.8 In the lung of patients with ILDs, TNF-αstimulates and regulates the synthesis and release of other lymphokines such as IL-1, GM-CSF, Platelet-activating factor, IL-6 and increases prostaglandin (PG) E2 production9 .

Sulfur Mustard (SM) induced cytotoxicity is due to the alkylation of a critical intracellular target, thereby interrupting the control of normal cellular processes.10 Recent studies have, nevertheless, reported characteristic clinical and immune responses to SM, which include a unique case definition of pulmonary damage and cytokine production.11-16 SM exposure produces a chronic immunocompromised condition, which systemically induces abnormal serum levels of Th1 and Th2 cytokines.17-19 Few studies have been carried out on in vitro models of respiratory epithelial cells, which represent one of the main targets of SM.20-22 A study on the effects of SM on tracheal epithelial cells has shown that mild doses of SM induce apoptosis, while higher doses induce necrosis.23 Evidence of an increased death toll from respiratory diseases and a higher incidence of chronic bronchitis have been reported in Japanese workers .24 Manning et al.25 found that pneumonia was the only cause of increased mortality among British SM factory workers. In vitro studies on human mononuclear leukocytes have demonstrated SM-induced mononuclear leukocyte cell death in a time-dependent fashion26. In a separate study Lardot et al.27 demonstrated that SM exposure can modify the expression by cultured human keratinocytes of interleukin-8. They observed a significant increase in the amount of IL-8 secretion by human keratinocyte treated with 1×10-3 M SM after 6 hours of exposure. They have also demonstrated that treatment with 1×10-6 M and 1×10-5M SM, induced no significant differences compared with the control group.27

On account of the high incidence of debilitating exposure to SM during the Iran-Iraq war, there is an increased interest in its mechanism of action and in the development of therapeutic interventions to prevent SMinduced lesions. The aim of this study was to investigate whether SM affects cytokine production by macrophages involved in lung inflammation.



Twenty-four male rats, aged 8 weeks old and weighing 150 g, were divided into two twelve-member groups: control and test. The animals were maintained in dust-free bedding cages in the animal unit. The twelve animals in the test group were exposed to the vapours of SM (obtained from The Ministry of Defence) through inhalation (42.3 mg/m3)28 for 30 minutes in a small cage. The animals in the control group were exposed to acetone only. During each two months, two animals were examined for lung damage29. Based on no clear damage to the lung after 2 and 4 months of exposure and death of two animals by six months29, the experiment was terminated at six months for cytokine assay in the remaining 6 animals of each group.

Serum Samples

After the animals had been anesthetised, blood samples were obtained from their heart using 5-mL syringes. The blood samples were centrifuged at 1500 rpm for 10 minutes, and sera were separated and kept at -20ºC until they had been analysed for cytokine assay using the ELISA kit (Bender Med Systems, USA).

Broncho Alveolar Lavage (BAL)

The animals having been anesthetised, their BAL was obtained by cannulating the trachea and lavaging the lung six times with a single volume of 15 mL of ice-cold sterile NaCl 0.9%. This BAL fluid (BALF) was centrifuged (1.000 ×g) for 10 minutes at 4ºC.

The free cells BAL fluid was kept at -20ºC until it had been analysed for cytokine assay using the ELISA kit (Bender Med Systems, USA). All cytokine assays was performed in molecular immunology laboratory in Baqiyatallah Medical Sciences University.

Statistical Analysis

All experiments were performed six times (n=6) and the data were analysed by Mynova software using t-test.


The results showed significant differences (p<0.001) between the control and exposed groups in terms of all cytokine (IL-9, γIFN, TGFβ, IL-6, IL-1β, IL-12 and TNFα) productions in both the BAL fluid and serum (Fig. 1,2). The most noticeable enhancement in cytokine release was seen in IL-9, which was 615.93% and 321.88% for the BAL fluid and serum, respectively (p<0.001). After IL-9 the highest increase was demonstrated for TGF-βand IL-6 in the BAL fluid which was 200.85% and 125.50% respectively. In both BAL and sera, a significant reduction was seen by γIFN (58.95% and 69.02% for BAL and sera respectively). The rate of reduction for IL-1β, IL-12, and TNF-α in the BAL was more than that in sera, but no significant difference was seen in the rate of TNF-α reduction in BAL and sera (Table I).


The reactivity of cytotoxic alkylating agents with DNA, RNA and proteins can cause mutagenic damage and cell death.30 Moreover, the underlying immunological effects of SM exposure have remained poorly defined. Recent studies, however, have reported clinical and immune responses to SM, which include a unique case definition of pulmonary damage and cytokine production. Previous studies indicate that SM exposure produces a chronic immunocompromised condition, which systemically induces abnormal serum level of Th1 and Th2 cytokines.17 Since infiltration by lymphocytes and polymorphonuclear leukocytes represents one of the first events observed in vivo upon exposure to SM, this study examined whether SM exposure could modify the production of cytokines.

The results of this study showed that SM inhalation up-regulated IL-6, IL-9 and TGF-β release in the BAL fluid and serum and down-regulated IFN-γ, IL-1β, IL-12, and TNF-α (p<0.001), indicating the possible role of cytokines in lung inflammation in chemical warfare victims.

In our study, a high level of IL-9, TGF-β and IL-6 were demonstrated; indicating the possible effect of SM induced lung injury via these above mentioned cytokines. Supporting our view, Arrovo et al.12 reported an increase in IL-1β, IL-6 and TNF-α release by human epidermal keratinocytes exposed to SM, demonstrating the important role of these cytokines in SM injury. In favour to our findings important roles for TNF-α, TGF-β in lung inflammation have been established by other researchers 31, 32

Aghanouri et al.33 reported a significant difference in the level of TGF-βbetween chemical warfare victims and control group, proposed that IFN-γ could be a useful drug for those suffering from lung inflammation. Hassan et al. 34 in a recent investigation demonstrated that SM caused an overall suppression of the immune response. There is also ample information demonstrating the role of IL-9, IL-12 and IFN-γ in promoting lung injury. 35, 36 In this study, the level of TGF-β and IL-9 were increased, while IFN-γ showed a significant reduction (p<0.001). Therefore it seems that macrophages by enhancing the IL9 production are trying to act in opposite action of TGF-β. Related to our results, Gary et al.36 demonstrated that IL-9 has the capacity to modulate the development of lung fibrosis. Noticeable changing in the level of cytokine after six months exposure to SM in this study, show that SM may induce an alteration in gene expression which are primarily involved in inflammation, apoptosis and cell cycle regulation.37-40 Related to our view, microarray analysis provided the opportunity to identify multiple transcriptional biomarkers associated with SM exposure.41 Rogers 41 demonstrated that in an SM-exposed skin, a total of 19 genes within apoptosis, transcription factors, cell cycle, inflammation, oncogens and tumor suppressors categories have been up-regulated.

In conclusion, in light of the results of this study and other similar investigations, it seems that SM, by modulating macrophage function in terms of the production of inflammatory cytokines, might be responsible for lung inflammation. The results of this study also provide a further understanding of the molecular responses to inhalation SM exposure, and enable the identification of potential diagnostic markers and therapeutic targets for treating SM injury.


Thanks are due to The Research Center of the Chemically Injured for financial assistance. This project was approved by the vice chancellor of research of Baqiyatallah Medical Sciences University.

  1. Atamas SP, White B: Cytokine regulation of pulmonary fibrosis in scleroderma. Cytokine and Growth Factor Reviews 14: 537-50, 2003.
  2. Semenzato G, Adami F, Maschio N, Agostini C, Immune mechanisms in interstitial lung disease. Allergy 55 (12): 1103-20, 2000.
  3. McLane MP, Haczku A, Van de Rijin M: Interleukin-9 promotes allergen-induced eosinophilic inflammation and airway hyperresponsivness in transgenic mice. Am J Respir Cell Mol Biol 19: 713-20, 1998.
  4. Agostini C, Trentin L, Perin A: Regulation of alveolar macrophage –T cell interactions during Th1-type sarcoid inflammatory process. Am J Physiol 277: L240-50, 1999.
  5. Mikuniya T, Nagai S, Shimoji T: Quantitative evaluation of the IL-1 beta and Il-1 receptor antagonist obtained from BALF macrophages in patients with interstitial lung diseases. Sarcoidosis Vasc Diffuse Lung Dis 14: 39-45, 1997.
  6. Tinkle SS, Newman LS: Beryllium-stimulated release of tumor necrosis factor-alpha, interleukin-6, and their soluble receptors in chronic beryllium disease. Am J Respir Cri Care Med 156: 1884-91, 1997.
  7. Moller DR: Cells and cytokines involved in the pathogenesis of sarcoidosis. Sarcoidosis Vac Diffuse Lung Dis 16: 24-31, 1999.
  8. Baughman RP, Lower EE, Miller MA, Bejarano PA, and Heffelfinger SC: Overexpression of transforming growth factor-alpha and epidermal growth factorreceptor in idiopatic pulmonary fibrosis. Sarcoidosis Vasc Diffuse Lung Dis 16: 57-61,1999.
  9. Bost TW, Riches DW, Schumacher B: Alveolar macrophages from patients with beryllium disease and sarcoidosis express increased levels of mRNA for tumor necrosis factor–alpha and interleukin-1 beta. Am J Respir Cell Mol Biol 10: 506-13, 1994.
  10. Papirmeister B, Feiter AJ, Robinson SI, Ford RD: Medical defence against mustard gas: Toxic Mechanisms and pharmacological implications. CRC Press, Boca Raton, FL. 11: 359, 1991.
  11. Cowan FM, Broomfield CA, Smith WJ: Suppression of sulfur mustard-increased IL-8 in human keratinocyte cell cultures by serine protease inhibitors: implications for toxicity and medical countermeasures. Cell Biol Toxicol 18(3): 175-80, 2002.
  12. Arrovo CM, Schafer RJ, Kurt EM, Broomfield CA, Carmichael AJ: Response of normal human keratinocytes to sulfur mustard: cytokine release. J Appl Toxicol 1:S63-72, 2000.
  13. Arrovo CM, Broomfield CA, Hackley BE Jr: The role of interleukin-6 in human sulfur mustard (HD) toxicology. Int J Toxicol 20(5):281-96, 2001.
  14. Arrovo CM, Burman DL, Kahler DW, et al: TNFalpha expression patterns as potential molecular biomarker for human skin cells expressed to vesicant chemical warfare agents: Sulfur mustard (HD) and Lewisites (L). Cell Biol Toxicol 20 (6): 345-59, 2004.
  15. Sabourin CL, Danne MM, Buxton KL, et al: Cytokine, chemokine, and matrix metalloproteinase response after sulfur mustard injury to weanling pig skin. J Biochem Mol Toxicol 16(6): 263-72, 2002.
  16. Ghanei M, Mokhtari M, Mohammad MM, Aslani J: Bronchiolitis obliterans following exposure to sulfur mustard: Chest high resolution computed tomography. Eur J radiol 52(2): 164-69, 2004.
  17. Rook G, Zumla A: Gulfwar syndrome: is it due to a systemic shift in cytokine balance towards a Th2 profile. Lancet 349: 1831-33, 1997.
  18. Anderson DR, Yourick JJ, Moeller RB, Petrali P, Young GD, Byers SL: Pathologic changes in rat lungs following acute sulfur mustard inhalation. Inhalation Toxicology 8: 285-97, 1996.
  19. Calvet JH, Jerreau PH, Levame M, d’Orto MP, Lorino H, Harf A, Macquin-Mavier I: Acute and chronic respiratory effects of sulfur mustard intoxication in guinea pig. Journal of Applied Physiology 76: 681-88, 1994.
  20. Chevillard M, Lainee P, Robineau P, Puchelle E: Toxic effects of sulfur mustard on respiratory epithelial cells in culture. Cell and Biological Toxicology 8:171-81, 1992.
  21. Lindsay CC, Hambrock JL: Protection of A 549 cells against the toxic effects of sulfur mustard by hexamethylenetetramine. Human and Experimental Toxicology 16:106-14, 1997.
  22. McClintock SD, Hoesel LM, Das SK, et al: Attenuation of half sulfur mustard gas-induced acute lung injury in rats. J Appl Toxicol 26(2):126-31,2006.
  23. Calvet JH, Feuermann M, Llorente B, Loison F, Harf A, Marano F: Comparative toxicity of sulfur mustard and nitrogen mustard on tracheal epithelial cells in primary culture. Toxicology in Vitro 13: 859-66, 1999.
  24. Sasser LB, Cushing JA, Dacre JC: Two generation reproduction study of sulfur mustard in rats. Reproductive Toxicology 10 (4): 311-319, 1996.
  25. Manning KP, Skegg DCG, Stelland PM, Doll R: Cancer of the larynx and other occupation hazards of mustard gas workers. Clin Otolaryngol 6:165-70,1981.
  26. Meir HL: The time-dependent effect of 2,2’dichlorodiethyl sulphide (sulfur mustard, HD, 1,1’–thiobis{ 2-chloroethanol} on the lymphocyte viability and the kinetics of protection by poly (ADP-ribose) polymerase inhibitors. Cell Biol Toxicol 12 (3):14753, 1996.
  27. Lardot C, Dubois V, Lison D: Sulfur mustard upregulates the expression of intereukin-8 in cultured human keratinocytes. Toxicology Letters 110: 29-33, 1999.
  28. Kumar O, Sugendran K., Ijayaraghavan R: Protective effect of various antioxidants on the toxicity of sulfur mustard administered to mice by inhalation or percutaneous routes. Chemio-Biological Interactions 134: 1-12, 2001.
  29. Ahmadi K, Akbari HMH: Effect of sulfur mustard on the lung tissue. J of Military Medicine 7 (3): 221-225, 2005.
  30. Sanderson BJS: Shield AJ: Mutagenic damage to mammalian cells by therapeutic alkylating agents. Mutation Research 355: 41-57, 1996.
  31. Gharaee-Kermani M, Phan SH: The role of eosinophils in pulmonary fibrosis. Int J Mol Med 1(1): 43-53, 1998.
  32. Huaux F, Lardot C, Delos M, et al: Lung fibrosis induced by silica particles in NMRI mice is associated with an upregulation of the p40 subunit of interleukin12 and Th2 manifestations. Am J of Respir Cell Mol Biol 20(4): 561-70, 1999.
  33. Aghanouri R, Ghanei M, Aslani J, Keivani-Amine H, Rastegar F, Karkhaneh A: Fibrogenic cytokine levels in bronchoalveolar lavage aspirates 15 years after exposure to sulfur mustard. Am J Physiol Lung Cell Mol Physio 287(6): L1160-64, 2004.
  34. Hassan ZM, Ebtekar M: Modeling for immunosuppression by sulfur mustard. Int Immunopharmacol 1(3): 605-10, 2001.
  35. Arras M, Huaux F, Vink A, et al: Interleukin-9 reduces lung fibrosis and type 2 immune polarization induced by silica particles in a murin model. Am J Respir Cell Mol Biol 24: 368-75, 2001.
  36. Hoyle GW, Arnold R: IL-9 and lung fibrosis: A Th2 good guy. Am J Respir Cell Mol Biol 24(4): 365-70, 2001.
  37. Sabourin CL, Petrali JP, Casillas RP: Alteration in inflammatory cytokine gene expression in sulfur mustard-exposed mouse skin. J Biochem Mol Toxicol 14(6): 291- 302, 2000.
  38. Sabourin CL, Rogers JV, Choi YW, et al: Time and dose dependent analysis expression using microarrays in sulfur mustard-exposed mice. J Biochem Mol Toxicol 18(6): 300-12, 2004.
  39. Ray R, Hauck S, Kramer R, Benton B: A convenient fluorometric method to study sulfur mustard-induced apoptosis in human epidermal keratinocytes monolayer microplate culture. Drug Chem Toxicol 28(1): 105-16, 2005.
  40. Rosental DS, Velena A, Chou FP, et al: Expression of dominant–negative Fas –associated death domain blocks human keratinocyte apoptosis and vesication induced by sulfur mustard. J Biol Chem 278(10): 8531-40, 2002.
  41. Rogers JV, Choi YW, Kiser RC, et al: Microarray analysis of gene expression in murin skin exposed to sulfur mustard. J Biochem Mol Toxicol 18(6):289-99, 2004.

Copyright 2006 -Medical Journal of the Islamic Republic of Iran

The following images related to this document are available:

Photo images

[mr06013f2.jpg] [mr06013f1.jpg] [mr06013t1.jpg]
Home Faq Resources Email Bioline
© Bioline International, 1989 - 2024, Site last up-dated on 01-Sep-2022.
Site created and maintained by the Reference Center on Environmental Information, CRIA, Brazil
System hosted by the Google Cloud Platform, GCP, Brazil