International Immunopharmacology 77 (2019) 105844 Contents lists available at ScienceDirect International Immunopharmacology journal homepage: www.elsevier.com/locate/intimp Tear and serum interleukin-8 and serum CX3CL1, CCL2 and CCL5 in sulfur T mustard eye-exposed patients ⁎ Tooba Ghazanfaria, ,1, Hassan Ghasemib, Roya Yaraeec,1, Mahmoud Mahmoudid, Mohammad Ali Javadie, Mohammad Reza Soroushf, Soghrat Faghihzadehg,2, Ali Mohammad Mohseni Majda,1, Raheleh Shakerih, Mahmoud Babaeii,j, Fatemeh Heidarya,1, Zuhair Mohammad Hassank a Immunoregulation Research Center, Shahed University, Tehran, Iran b Department of Ophthalmology, Shahed University, 3319118651 Tehran, Iran c Department of Immunology and Immunoregulation Research Center, Shahed University, Tehran, Iran d Immunology Research Center, Mashhad University of Medical Sciences, 9138813944 Mashhad, Iran e Ophthalmic Research Center, Shahid Beheshti University of Medical Sciences, 1983969411 Tehran, Iran f Janbazan Medical and Engineering Research Center (JMERC), NO.17, Farrokh St., Moghaddas Ardebily Ave., Chamran Highway, 1985946531 Tehran, Iran g Department of Biostatistics and Social Medicine, Zanjan University of Medical Sciences, Zanjan, Iran h Department of Biological Science and Biotechnology, Faculty of Science, 6617715175, University of Kurdistan, Sanandaj, Iran i Department of Ophthalmology, Baqiyatallah University of Medical Sciences, 1435916471 Tehran, Iran j Trauma Research Center, Baqiyatallah University of Medical Sciences, Tehran, Iran k Department of Immunology, School of Medical Sciences, Tarbiat Modares University, 14115111 Tehran, Iran ARTICLE INFO ABSTRACT Keywords: Background: The serum and tear levels of four inflammatory chemokines were evaluated in sulfur mustard (SM)- Sulfur mustard exposed with serious ocular problems. Ocular injury Materials and methods: In this study, 128 SM-exposed patients and 31 healthy control participants participated. MCP-1/CCL2 Tear and serum levels of chemokines were assessed by ELISA method. RANTES/CCL5 Results: There was no significant difference in the serum level of IL-8/CXCL8, CX3CL1/fractalkine, CCL2/MCP- IL-8/CXCL8 1, and CCL5/RANTES between all SM-exposed subjects and control groups. The tear level of IL-8 in the SM- CX3CL1/fractalkine exposed group was lower than the control group, but the difference was not significant. In the SM-exposed group with the abnormalities in tear breakup time (TBUT) test, fundus and pannus formation were significantly higher than SM-exposed patients without these problems. CX3CL1 levels have significantly increased in SM-exposed group with blepharitis, pterygium, and conjunctival pigmentation as compared with the control group. Besides, significantly higher levels of CX3CL1 were observed in SM-exposed group with or without bulbar conjunctival hyperemia and abnormal vessels a well as with fundus abnormality compared to the control group. Only, SM- exposed group with subconjunctival fibrosis had significantly lower levels of CCL5 than SM-exposed group without this problem. Conclusion: The higher level of CX3CL1 and consistent levels of IL-8/CXCL8, MCP-1/CCL2, and RANTES/CCL5 in SM-exposed individuals may indicate an anti-inflammatory response against the destructive effects of SMgas. High tear level of IL-8/CXCL8 reflects the severity of ocular surface abnormalities, yet significantly low tearlevel found in mild SM-exposed subgroup compared with the control group. The lower levels of CX3CL1 and RANTES/ CCL5 may represent the different pathophysiology which requires further studies. ⁎ Corresponding author at: Immunoregulation Research Center and Dep of Immunology, Shahed University, Tehran, Iran. E-mail addresses: [email protected] (T. Ghazanfari), [email protected] (M. Mahmoudi), [email protected] (M. Babaei), [email protected] (Z.M. Hassan). 1 Department of Immunology, Shahed University, Tehran, Iran, 3319118651. 2 Faculty of Medicine, Zanjan University of Medical Science, Zanjan, Iran, 4515613191. https://doi.org/10.1016/j.intimp.2019.105844 Received 20 May 2019; Received in revised form 17 August 2019; Accepted 19 August 2019 Available online 24 October 2019 1567-5769/ © 2019 Published by Elsevier B.V. T. Ghazanfari, et al. International Immunopharmacology 77 (2019) 105844 1. Introduction punctal abnormality), tear status (tear break up time [TBUT] test and tear meniscus height), bulbar conjunctiva (limbal ischemia, hyperemia, Sulfur mustard (SM) is a chemical warfare agent used against the abnormal vessels, pterygium and subconjunctival fibrosis), limbal tissue Iranian army and civilians by Iraqi military forces from 1980 to 1988 (pigmentation, abnormal vessels and pannus), cornea (calcium de- [1]. The main targets of SM are the eyes, skin, and respiratory system position, melting, verticillata, vascularization, epithelial, stromal, and [2]. Varying degrees of ocular abnormalities, including chronic ble- endothelial abnormalities), lens (nuclear sclerotic [NS], cortical [C] and pharitis, meibomian gland dysfunction (MGD), dry eye, limbal posterior subcapsular [PS] cataract) and fundus. Evaluation of the ischemia, limbal stem cell deficiency, aberrant conjunctival vessels, posterior segment was carried out using direct and indirect ophthal- corneal neovascularization; and secondary degenerative changes, like moscopy (HEINE, Germany). Final ophthalmologic assessments were lipid and amyloid deposition, corneal irregularity, thinning and scar- categorized into normal, mild, moderate, and severe, based on the chart ring has been reported in 75% to 90% of SM-exposed individuals [3]. of the War Veterans Foundation (Iranian Ophthalmic Committee of Photophobia, ocular surface discomfort (burning, itching, and redness), Chemical Warfare Veterans) [10]. Demographic and clinical data of the bulbar conjunctival abnormalities, and limbal tissue changes were re- studied group were described in our previous study [11]. ported by Ghasemi et al. among SM-exposed victims in Sardasht-Iran cohort study (SICS) [4]. In the previous study, we have shown altera- 2.2. Serum preparation tions in the serum levels of chemokines in Sardasht City residents 20 years after SM exposure [5]. In addition, we found a positive asso- For serum preparation, we have drawn blood samples of all subjects ciation between serum levels of proinflammatory cytokines (interleukin which after clotting, the sera were separated, aliquoted, and kept at [IL]-1β, IL-8, IL-12 and RANTES [regulated on activation, normal T cell −80 °C. expressed and secreted]) and acne among SM-exposed population in Sardasht City, Iran [6]. 2.3. Tear collection Chemokines are a large family of cytokines produced during in- flammatory reactions. They participate not only in the regulation of One drop (200 μL) of sterile 5% sodium chloride instilled in the inflammation and various immune responses but also organ develop- inferior fornices of both eyes to avoid any ocular surface trauma from ment, angiogenesis/angiostasis, leukocyte trafficking and homing, tu- capillary or microtubes. The available basal and reflex tears samples morigenesis, and metastasis. Chemokines are classified into four major collected via the placement of a sterile Weck-Cel sponge in the lacrimal groups of CXC (α chemokines), CC (β chemokines), C (γ chemokines), lake for a few minutes from 118 veterans and 20 controls, immediately and CX3C (δ chemokines) [7]. The CC and CXC chemokines are che- [12]. Dilution of tear samples conducted in special tubes using 200 μL motactic for monocytes and neutrophils, respectively [5]. The expres- of RPMI medium was and afterward, centrifuged at 2000 ×g for 20 min sion of CX3CL1 (called also fractalkine) has been reported in a variety to isolate the tear fluid from the Weck-Cel sponges. Samples stored at of ocular tissues and cells which is thought to be involved in regulating −80 °C pending laboratory measurements. leukocyte migration in inflammatory eye diseases such as anterior uveitis and retinochoroiditis [8]. The contribution of chemokines such 2.4. Chemokine measurement as chemokine (C-X-C motif) ligand 8 (CXCL8) or interleukin (IL)-8, CXCL10/IP-10 (interferon g-inducible protein-10), chemokine (CeC Four chemokines were selected because they represent markers motif) ligand 2 (CCL2) or monocyte chemoattractant protein 1 (MCP- implicated in infectious disease pathobiology, including inflammation 1), chemokine (CeC motif) ligand 5 (CCL5) or RANTES (regulated on and endothelial activation. The serum concentration of chemokines (IL- activation, normal T cell expressed and secreted), and chemokine (CeC 8/CXCL8, CX3CL1/fractalkine, CCL2/MCP-1, and CCL5/RANTES) in motif) ligand 4 (CCL4) or macrophage inflammatory protein-1 beta SM-exposed and control groups were measured using Human DuoSet® (MIP-1b) in eye inflammation has been already investigated [9] ELISA Development kits (R&D Systems, US) according to the manu- whereas their involvement in ocular abnormalities in response to SM facturers' instructions as described previously [13]. We validated ELISA exposure has not been studied. To evaluate eye injuries of SM exposure, kits before use, and we obtained appropriate samples dilutions for each we measured the serum and tear levels of chemokines in the Iranian biomarker by testing a dilution curve of serum received from subjects. war veterans exposed to SM with ocular abnormalities. Moreover, as- Chemokine concentrations (pg/mL) were deduced from the equivalent sessing the involvement of chemokines in SM-exposed individuals with standard
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