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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 2  |  Issue : 4  |  Page : 306-310

Determination of CD4, CD8, and IL-8 levels in serum and bronchoalveolar lavage fluid of anthracosis patients


1 Tracheal Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
2 Department of Pulmonology, Qom University of Medical Sciences, Qom, Iran
3 Chronic Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran
4 Clinical Tuberculosis and Epidemiology Research Center, National Research Institute for Tuberculosis and Lung Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran

Date of Submission15-Aug-2018
Date of Decision30-Aug-2018
Date of Acceptance05-Sep-2018
Date of Web Publication11-Dec-2018

Correspondence Address:
Dr. Atefeh Abedini
Chronic Respiratory Diseases Research Center, National Research Institute of Tuberculosis and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran
Iran
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/bbrj.bbrj_99_18

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  Abstract 


Background: Anthracosis is a chronic pulmonary disease with black pigmentation of the bronchial mucosa, caused by carbon accumulation in the lungs, and in some cases, associated with pulmonary tuberculosis (TB). The purpose of this study is to evaluate the serum and bronchoalveolar lavage (BAL) fluid levels of the CD4/CD8 ratio and interleukin 8 (IL-8), as the potential diagnosis and prognosis biomarkers for anthracosis. Methods: Of 60 patients, referred to the Masih Daneshvari Hospital, 30 anthracosis patients, confirmed by bronchoscopy, were included as the case group. BAL sample and blood samples were collected from all individuals and sent to the immunology laboratory. Patients with no anthracosis on bronchoscopy, and suspected to have TB, were included in the control group. In addition, BAL samples were used for BK-polymerase chain reaction. Results: In this case–control study, 30 anthracosis patients and 30 controls were investigated. There were no significant differences in IL-8 of patients with anthracosis, compared to others. There were significant differences of the CD4/CD8 ratio in BAL fluid, between anthracosis cases and the control group (1.01 ± 0.77 vs. 2.41 ± 3.50; P = 0.04), and significant differences were seen in serum levels of two groups (P = 0.02). The rate of the confirmed pulmonary TB which was 88.9% in patients with anthracosis was significantly higher than the control group. Conclusion: These outcomes suggest that changes in the CD4/C8 levels in serum and BAL fluid may have a leading role in the diagnosis or prognosis of anthracosis. In addition, due to the strong association of anthracosis and pulmonary TB, TB should be considered in patients with anthracosis, which in turn can eventuate to the early distinguish and cure of the patients.

Keywords: Anthracosis (D055008), biomarkers (D015415), bronchoalveolar lavage (D018893), interleukins (D007378), tuberculosis (D014376)


How to cite this article:
Kiani A, Ramazanpour M, Razavi F, Jamaati H, Mortaz E, Abedini A. Determination of CD4, CD8, and IL-8 levels in serum and bronchoalveolar lavage fluid of anthracosis patients. Biomed Biotechnol Res J 2018;2:306-10

How to cite this URL:
Kiani A, Ramazanpour M, Razavi F, Jamaati H, Mortaz E, Abedini A. Determination of CD4, CD8, and IL-8 levels in serum and bronchoalveolar lavage fluid of anthracosis patients. Biomed Biotechnol Res J [serial online] 2018 [cited 2019 Jan 16];2:306-10. Available from: http://www.bmbtrj.org/text.asp?2018/2/4/306/247249




  Introduction Top


Anthracosis is a chronic pulmonary disease, usually specified by clinical symptoms such as a cough and shortness of breath, without a history of smoking. This disease can be determined as a superficial black discoloration of bronchi which is due to the carbon accumulation in lungs, aspiration of coal dust, or frequent contact with the air pollution.[1] In addition, it may be characterized as a scattered focus of black spots.[2],[3]

Histopathology of the lung tissue shows carbon and free particles, inside the macrophage cytoplasm of the bronchial wall and in the mediastinal lymph nodes.[4] It may be diagnosed in different radiological findings, including chest X-ray or computed tomography (CT). However, the most gold standard method for diagnosing anthracosis is bronchoscopy.[5] Nonetheless, the above-mentioned methods are aggressive and have high costs for patients. Therefore, finding easier, safer, and inexpensive diagnostic methods, with a high sensitivity for evaluation of changes of biomarkers for early diagnosis and prognosis, seems to be necessary. Examining immune factors, such as cytokines and CD markers (e.g., CD4 and CD8) can be considered as options for diagnosis, and ultimately treatment of this disease. Cytokines are extracellular signaling proteins that regulate fibrotic and inflammatory responses. The epithelial cells, endothelial cells, fibroblasts, and inflammatory cells are the main sources of cytokines in the lungs.[6] The cytokines of serum, including interleukin 8 (IL-8) are considered as biomarkers for the progression of pneumoconiosis.[7],[8] Analysis of inflammatory cell count and the CD4/CD8 ratio in bronchoalveolar lavage (BAL) and serum provides particular diagnostic evidence, in some pulmonary diseases, such as sarcoidosis and eosinophilic pneumonia.[9],[10]

Although many studies have been performed on pneumoconiosis disease, the evaluation of immune parameters for the pathogenesis of anthracosis is still unknown and only a few studies are available. Therefore, this study was aimed to evaluate the changes in immune factors, in anthracosis patients, in order to find a potential biomarker for earlier diagnosis. On the other hand, BK-polymerase chain reaction (BK-PCR) was also used to evaluate the association between tuberculosis (TB) and anthracosis.


  Methods Top


Subjects

The current case–control study conducted on 30 anthracosis patients, confirmed by bronchoscopy, and 30 suspected TB patients, with no anthracosis. Serum and BAL samples of all individuals were taken and then referred to the immunology laboratory for more analysis. The study was approved by NRITLD Ethics Committee and the written informed consent was obtained from each participant The Ethical committee approval number was IR.SBUM.NRITLD.REC.1396.544 dated 2016-2018.

Blood collection and biochemical assays

On the same day of bronchoscopy, we collected the blood samples of all individuals. Four to five milliliters of blood was collected from each participant and then centrifuged at 3000 rpm for 15 min at 4°C. The samples were taken at −80°C, for further analyses.

Processing of bronchoalveolar lavage fluid and differential cell counts

The BAL fluid of all participants was collected under local anesthesia, using fibrotic bronchoscopy before treatment. The BAL fluid samples were analyzed for total and differential cell counts.

The BAL was centrifuged at 400 g for 10 min at 4°C, in order to get supernatant. The BAL fluid samples which were cell-free were taken at − 80°C, for further analyses. The specimens were frozen about 30 min after the procedure of bronchoscopy. In order to gain a cell pellet, the BAL was passed through the sterile gauze and was then centrifuged. The pellet of cells was washed with 50 ml of Ca2+/Mg2+ free Hanks' balanced salt solution and then incubated for 20 min with 10% bovine serum albumin (Gibco). The cells were enumerated in a hemocytometer slide (using Kimura counterstain). The viability of cells was determined using trypan blue exclusion. In order to get differential cell counts, cytospins were conducted using 10 cells in each slide and were stained with May–Grunwald–Giemsa. After that, we added the pairs of monoclonal antibodies to CD4 and CD8 lymphocytes then we incubated it for 30 min. Subsequently, we performed flow cytometry.

Flow cytometry

This method was conducted using FACS Calibur (Becton Dickinson, San Jose, CA, USA). Based on the number of cells obtained, cells were stained at room temperature for 30 min with 100 ml of fluorescein isothiocyanate-labeled anti-CD4 and PE-anti-CD8 monoclonal antibodies then washed once in FACS buffer. The surface expression of CD4 and CD8 were defined by the flow cytometry. Lymphocytes were defined by forward and side scatter parameter, and they were gated to include cells within the windows set. Flow cytometry data were analyzed within 24 h after staining, using (flow Joe) software. The outcomes are expressed, as the percentage of positively labeled CD4 and CD8.

Cytokines enzyme-linked immunosorbent assay

The BAL levels of IL-8 were measured by the enzyme-linked immunosorbent assay kits (R & D system, London, UK), based on the manufacturer's instructions.

BK-polymerase chain reaction

The BAL samples were used for BK-PCR and methods have been down as described earlier.[11] The amplicor system was utilized, using biotinylated specific primers to create a specific DNA sequence of 584 bp from mycobacterium TB. The test was carried out in accordance with the manufacturer's instructions. One positive proliferation control and three negative controls were used in all runs. The results of the controls were presented, based on criteria, set by the producer.

Statistical analyses

All data were analyzed, under the usage of statistical software (IBM Corp. Released 2011. IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp). The CD4/CD8 ratio and IL-8 were analyzed, using independent t-test, and for BK-PCR, using Fisher test. Mean ± standard deviation was used for data description. P < 0.05 was established statistically significant.


  Results Top


In this study, 60 participants, including 29 men and 31 women with the mean age of 59.28 ± 15.60 years were investigated. They were classified into two groups. The control ones composed of 30 suspected TB patients, having three negative sputum sample test and a definite diagnosis of not having anthracosis, and the patient group, composed of 30 individuals with a bronchoscopic anthracosis diagnosis.

Data regarding CD4 and CD8 in serum and BAL fluid, serum IL8 level and CD4/CD8 ratio in BAL, and serum of anthracosis patients are summarized in [Table 1]. These results do not show any significant differences between males and females.
Table 1: The levels of CD4, CD8, and their ratio in bronchoalveolar lavage fluid and serum and serum interleukin-8 level in anthracite patients, based on the gender

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The CD4/CD8 ratio in BAL fluid [Figure 1], obtained from anthracosis patients was significantly lower than others (1.01 ± 0.77 vs. 2.41 ± 3.50; P = 0.04). In addition, as shown in [Figure 1], the CD4/CD8 ratio in the serum of anthracosis patients was significantly higher than others (1.28 ± 0.69 vs. 0.86 ± 0.65; P = 0.02).
Figure 1: The CD4/CD8 ratio in bronchoalveolar lavage fluid and serum analyzed by flow cytometry. Statistical significance was established at *P < 0.05

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The comparison between the level of IL-8 [Figure 2] in the patients (11.22 ± 13.89) and controls (6.87 ± 11.68) did not show a significant difference.
Figure 2: Interleukin 8 in bronchoalveolar lavage fluid of anthracosis patients and controls

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[Table 2] summarizes the analysis of BK-PCR in all patients. According to Fisher's exact test and sig = 0.026, the difference in the positive values of BK-PCR in BAL fluid, between the two groups of anthracosis and control is statistically significant.
Table 2: BK-polymerase chain reaction of bronchoalveolar lavage fluid from anthracosis patients and the control group

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  Discussion Top


This study was aimed to investigate the changes in CD4, CD8, and IL-8 factors in serum and BAL fluid, in cases with pulmonary anthracosis, in an Iranian population. Anthracosis can be caused with the aspiration of dust, coal particles, and air pollution, characterized by the black discoloration of the airway mucosa. Our study showed that there were significant differences in BAL and serum levels of the CD4/CD8, between the two groups of anthracosis and control. That is while there were not any significant differences between IL-8 in the BAL fluids, from anthracosis patients and control groups.

Detection of cellular compositions (CD markers, such as CD8 and CD4) in serum and BAL fluid may help to understand the relevant pathophysiological mechanism and functional status of the cells in this disease. The analysis of changes in inflammatory cells and also the CD4/CD8 ratios in the BAL fluid and serum provides valuable diagnostic tools in some unusual pulmonary diseases, including hypersensitivity pneumonitis, sarcoidosis, and eosinophilic pneumonia.[10] Through a study of 261 patients consisted of 119 sarcoidosis and 142 nonsarcoidosis ILDs, Tanriverdi et al. showed that the mean CD4/CD8 ratio among sarcoidosis patients was significantly more than nonsarcoidosis ILDs. Therefore, between flow cytometric parameters of the BAL fluids, the CD4/CD8 may be useful for the diagnosis of sarcoidosis from other ILDs but not specific for other lung diseases.[12] Suzuki et al. through a study on 107 individuals who had diffuse lung diseases, showed that the CD4/CD8 ratio in BAL fluid was significantly more in sarcoidosis patients in comparison with patients with CT-ILD, IPF, and healthy individuals.[13] In contrast, Mota et al. reported a significant increase of lymphocytes in the BAL of nonsarcoidosis cases, compared to the patients with sarcoidosis.[14]

In this study, the CD4/CD8 ratio was examined. Based on our knowledge, this is the first evaluation of CD4/CD8 in the serum and BAL fluid of anthracosis patients. Our data showed statistically significant differences in the CD4/CD8 ratio in serum and BAL fluid of anthracosis cases, compared to the controls.

IL8 is considered, as a potent factor in cellular inflammation.[15] IL-8 is known as a significant activator as well as a chemical absorbent for neutrophils and also an important chemokine in inflammation of the lungs, caused by crystalline silica.[16],[17] IL-8 levels have been reported in monocytes supernatant, motivated spontaneously or with dust.[18] Lee et al. conducted a study on 110 coal mines in 2010, based on their findings IL-8 levels were significantly more than their control group, and also related to the degree of pneumoconiosis controls.[19] In 2014, Lee et al. also conducted a study on coal mine workers with pneumoconiosis and reported that the IL-8 levels in people with pneumoconiosis were significantly higher, compared to the controls.[15]

To the best of our knowledge, there is not any study, about the changes in IL-8 levels of anthracosis patients. In our present study, for the first time, changes in serum and BAL fluid of IL-8 levels were examined among patients with anthracosis. The results showed that IL-8 levels in BAL fluid of patients were not significantly different compared to the control subjects.

In our study, BK-PCR was also used to examine the association of TB with anthracosis. Based on the current hypothesis, some patients with anthracosis were not in contact with dust or soot, a significant percentage of them have had active pulmonary TB. The mechanism of action is in such a way that the anthracotic materials from the lymph nodes of patients with TB, located adjacent to the bronchial tubes, gradually discharged into the bronchi and will become a source of the anthracitic pigments inside the bronchi.[15],[20] In a study in South Korea in 1995, 908 cases that underwent bronchoscopy had been studied. Anthracofibrosis was diagnosed in 28 patients, of which, 17 (67%) had active pulmonary TB.[21] According to a study in 2003 in France, the presence of anthracotic plaque was introduced as a sign of the history of pulmonary TB with occupational pulmonary diseases and exposure to dust. In this article, it was demonstrated that the presence of pulmonary anthracosis is responsible for increasing the accumulation of fungi, as well as provide conditions for the growth of pathogens, including Mycobacterium TB.[22]

In our present study, using BAL fluid, the prevalence of TB and anthracosis was examined. Our outcomes revealed that of 30 anthracosis patients, 88.9% had positive BK-PCR of BAL fluid, and a significant difference was seen between the case and the control group (this rate was 11.1%). These results are consistent with the above-mentioned studies.


  Conclusions Top


Our data indicate that the CD4/CD8 ratio in serum and BAL fluid may have a role in the diagnosis of anthracosis. However, this disease cannot be diagnosed by the levels of IL-8.

Furthermore, because of the strong association of anthracosis with pulmonary TB in the present study, it can be concluded that the presence of anthracosis can be one of the strongest signs for the presence of pulmonary TB.

The most important limitation of this study was the absence of differentiation of the anthracosis subgroups, such as fibrotic anthracosis, bronchial anthracosis, and anthracnosis. In addition, the influence of genetics and ethnicity has not been considered. Therefore, a prospective study with a larger sample size and a distinction between type and degree of the disease is suggested, in order to obtain a more accurate interpretation of the association between immune factors and anthracosis disease.

On the other hand, due to the association of anthracosis with lung TB, precise preclinical studies and fiberoptic bronchoscopes are recommended for the diagnosis of pulmonary TB in these cases.

Acknowledgment

The authors wish to thank all the anthracosis patients who helped us to conduct this study and also we appreciate National Research Institute of TB and Lung Diseases (NRITLD), Shahid Beheshti University of Medical Sciences, Tehran, Iran, for their support.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Jamaati H, Bahrami N, Tabarsi P, Khosravi A, Kiani A, Abedini A, et al. Multi-gene expression in anthracosis of the lungs as one of the risk factors for non-small cell lung cancer Asian Pac J Cancer Prev 2017;18:3129-33.  Back to cited text no. 1
    
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Lee J, Kim YK, Seo YY, Choi EK, Lee DS, Kim YS, et al. Clinical characteristics of false-positive lymph node on chest CT or PET-CT confirmed by endobronchial ultrasound-guided transbronchial needle aspiration in lung cancer. Tuberc Respir Dis (Seoul) 2018;81:339-46.  Back to cited text no. 4
    
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Nario RC, Hubbard AK. Silica exposure increases expression of pulmonary intercellular adhesion molecule-1 (ICAM-1) in C57Bl/6 mice. J Toxicol Environ Health 1996;49:599-617.  Back to cited text no. 7
    
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Suzuki E, Tsukada H, Ishida T, Ishizuka O, Hasegawa T, Gejyo F, et al. Correlation between the numbers of gammadelta T cells and CD4+ HLA-DR+ T cells in broncho-alveolar lavage fluid from patients with diffuse lung disease. Tohoku J Exp Med 2002;196:231-40.  Back to cited text no. 13
    
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Singh V, Meena H, Bairwa R, Singh S, Sharma BB, Singh A, et al. Clinico-radiological profile and risk factors in patients with anthracosis. Lung India 2015;32:102-6.  Back to cited text no. 20
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21.
Chung MP, Lee KS, Han J, Kim H, Rhee CH, Han YC, et al. Bronchial stenosis due to anthracofibrosis. Chest 1998;113:344-50.  Back to cited text no. 21
    
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