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ORIGINAL ARTICLE
Year : 2019  |  Volume : 32  |  Issue : 3  |  Page : 961-966

Study of CD14+ and CD16+ peripheral blood monocytes in asthmatic patients


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Chest Diseases and Tuberculosis, Faculty of Medicine, Menoufia University, Menoufia, Egypt
3 Department of Clinical Pathology, Menouf Hospital, Menoufia, Egypt

Date of Submission08-Nov-2017
Date of Acceptance13-Dec-2017
Date of Web Publication17-Oct-2019

Correspondence Address:
Mona A Ibrahim
El-Bajor, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_735_17

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  Abstract 

Objective
This study aimed to investigate the enhanced frequencies of CD14++ CD16 + peripheral blood monocytes in asthmatic patients.
Background
CD16 + monocytes are increased in inflammatory conditions. It was reported that CD16 + monocytes can be divided into two subsets with different potential of modulating inflammatory responses: CD14++ CD16 + and CD14 + CD16 + monocytes. CD14++ CD16 + and CD14 + CD16 + monocyte subsets are quantified in asthmatic patients regarding severity of disease and glucocorticoids treatment options.
Patients and methods
CD14++ CD16 + was examined in 20 cases of completely controlled asthma, 20 cases of partly controlled asthma, 20 cases of uncontrolled asthma, and 25 healthy controls using flow cytometry.
Results
The results showed highly significant difference among the controlled asthma, partly controlled asthma, uncontrolled asthma, and control groups regarding pulmonary function tests and also showed highly significant difference among controlled asthma, partly controlled asthma, uncontrolled asthma, and control groups regarding percentage of CD14++ CD16 + cells. Moreover, the results showed highly significant correlation between CD14++ CD16 + cells and percentage of forced expiratory volume in 1 s, forced expiratory volume in 1 s/forced vital capacity, C-reactive protein, erythrocyte sedimentation rate, immunoglobulin E, neutrophils, and eosinophils (laboratory investigations) of controlled asthma, partly controlled asthma, uncontrolled asthma, and control groups.
Conclusion
CD14++ CD16 + monocytes showed the highest levels of expression in uncontrolled asthmatic cases as compared with other monocyte subsets. CD14++ CD16 + monocytes showed higher levels of expression in uncontrolled asthma cases than other asthmatic cases.

Keywords: asthma, CD14, CD16, flowcytometry, monocyte subsets


How to cite this article:
Montaser LM, Sonbol AA, Agha MA, Ibrahim MA. Study of CD14+ and CD16+ peripheral blood monocytes in asthmatic patients. Menoufia Med J 2019;32:961-6

How to cite this URL:
Montaser LM, Sonbol AA, Agha MA, Ibrahim MA. Study of CD14+ and CD16+ peripheral blood monocytes in asthmatic patients. Menoufia Med J [serial online] 2019 [cited 2019 Nov 12];32:961-6. Available from: http://www.mmj.eg.net/text.asp?2019/32/3/961/268841




  Introduction Top


Asthma is a common chronic respiratory disease affecting 1–18% of the population in different countries. Asthma is characterized by variable symptoms of wheeze, shortness of breath, chest tightness, and/or cough, and by variable expiratory airflow limitation. Both symptoms and airflow limitation characteristically vary over time and in intensity. These variations are often triggered by factors such as exercise, allergen or irritant exposure, change in weather, or viral respiratory infections [1]. Asthma diagnosis is usually based on the pattern of symptoms, response to therapy over time, and spirometry [2]. Asthma is clinically classified according to the frequency of symptoms, forced expiratory volume in 1 s (FEV1), and peak expiratory flow rate [3].

Monocyte is a subtype of leukocytes (white blood cells) being produced from hematopoietic stem cell precursors in the bone marrow, playing multiple roles in immune function [4]. Monocytes are a circulating population of mononuclear phagocytes that can be rapidly recruited to tissues in inflammation. Monocyte contribution to the immune response is tissue specific. This is largely owing to the response of monocytes to environmental triggers. Monocytes are important constituents of the innate immune system [5]. Monocytes are comprised of subsets characterized by different levels of expression of CD14 and CD16 molecules. 'Classical' monocytes are strongly positive for CD14 and negative for CD16 (the CD14++ CD16 − monocytes) and constitute the majority of all monocytes in healthy persons. In contrast, monocytes positive for CD16 account for only 5–15% of all monocytes, but their frequency increases significantly in inflammatory conditions such as sepsis, tuberculosis, and atherosclerosis [6]. The nonclassical monocyte showed low level of expression of CD14 and additional coexpression of the CD16 receptor (CD14+ CD16++ monocyte). The intermediate monocyte showed high level of expression of CD14 and low level of expression of CD16 (CD14++ CD16+ monocytes). The level of CD14 expression can be used to differentiate nonclassical and intermediate monocytes [7]. The frequencies of expression of CD14++ CD16+ monocytes were significantly higher in uncontrolled asthmatics as compared with healthy controls and controlled and partly controlled asthmatics. Skrzeczyńska et al. [8] showed that glucocorticoid treatment leads to decrease of CD14+ CD16+ monocytes in severe asthmatics. This is explained by the effects of long-term treatment with moderate to high doses of inhaled glucocorticoids. The aim of this work was to evaluate the frequencies of CD14++ CD16+ peripheral blood monocytes in asthmatic patients.


  Patients and Methods Top


This was a hospital-based, case–control (retrospective) study. The study was performed on 85 participants, involving 60 patients and 25 healthy age-matched and sex-matched controls. Their ages ranged between 16 and 80 years. They were randomly selected from Menoufia University Hospitals.

Exclusion criteria

Patients with other diseases such as, chronic obstructive pulmonary disease, pulmonary tuberculosis, sarcoidosis, bronchogenic carcinoma, and weight loss, and those taking nonsteroidal anti-inflammatory drugs, methotrexate, sulfasalazine, and immunosuppressive drugs were excluded from the study.

The study was approved by the ethics committee of our medical faculty, and written informed consents were taken from all participants before the study.

The participants in this study were classified into four groups:

  1. Group I: it included 25 apparently healthy individuals as a control group. They were age and sex matched with patient group. There were 13 males and 12 females, and their ages ranged between 19 and 70 years
  2. Group II: it included 20 patients with completely controlled asthma who had no treatment with inhaled steroids. There were seven males and 13 females, and their ages ranged between 29 and 55 years
  3. Group III: it included 20 patients with partly controlled asthma who had treatment with inhaled steroids. There were eight males and 12 females, and their ages ranged between 16 and 76 years
  4. Group IV: it included 20 patients with uncontrolled asthma who had treatment with inhaled steroids. There were 10 males and 10 females, and their ages ranged between 29 and 80 years.


For all participants, the followings were done: a venous blood sample of 8 ml was withdrawn from each individual under aseptic condition using sterile disposable syringes and then dispensed into four tubes: 2 ml of blood was delivered into EDTA containing tube for complete blood count and CD14 and CD16 monocytes analysis, 1.6 ml of venous blood was delivered in a vacutainer plastic tube containing citrate for determination of erythrocyte sedimentation rate, and 4 ml of blood was delivered into a plain tube, from which serum was separated and used for assessment of liver function tests, kidney function tests, C-reactive protein (CRP), and immunoglobulin E (IgE).

Complete blood count

Complete blood count was done by Sysmex XN1000 (Sysmex Company, Kobe, Japan), which is an automated blood counter. erythrocyte sedimentation rate procedure was done by manual Westergren method. Liver function tests and kidney function tests were done by Beckman AU480 Automated Chemistry Analyzer (Beckman, Carlsbad, California, USA).

C-reactive protein

CRP measurement was done by nephelometry (Specific Protein Analyzer; Heales, Shenzhen, China).

Immunoglobulin E

IgE measurement was done by enzyme-linked immunosorbent assay. IgE was done by vidas total IgE (Biomérievy, marcy I'Etoile, Lyon, France). It is an automated enzyme-linked fluorescent immunoassay based on one-step immunoassay sandwich method with a final fluorescent detection step for the quantitative measurement of IgE.

Analysis of CD14 and CD16 level was done by flow cytometry. The reagents used are PBS, provided by Sigma (Setagaya City, Tokyo, Japan) and stored at 4°C, and monoclonal antibodies, fluorescein isothiocyanate (FITC) conjugated mouse monoclonal antihuman antibodies against CD14 and phycoerythrin (PE) conjugated mouse monoclonal antihuman antibodies against CD16 (eBioscience, San Diego, California, USA).

Intended use of monoclonal antibodies was designed to quantitatively determine the percentage of cells bearing CD14 and CD16 within a population and qualitatively determine the density of CD14 and CD16 on cell surfaces by flow cytometry. Whole blood samples were collected in sterile tubes containing EDTA. Overall, 2 ml of ficoll solution was placed in a centrifuge tube and layered on top with 1 ml of blood sample, ensuring that the blood and ficoll solution do not mix, and centrifuged at 1800 rpm for 20 mm. The mononuclear layer was taken in a separate tube. The cell suspension was washed three times with PBS and centrifuged at 3000 rpm for 5 min. For each sample, two tubes were prepared: one for the test and the other for the unstained auto control. Overall, 100 μl of cells suspension in PBS was mixed with 10 μl FITC-conjugated anti-CD14 abs (mouse antihuman IgGl, κ, clone 61D3) and 10 μl PE-conjugated mouse monoclonal CD16 (clone B73.1, mouse antihuman IgG, κ) in the test tube. The mixture was incubated at 2–8°C for 30 min. The cells were washed twice in 2 ml PBS. The control contains 100 μl of cells without any stain. Finally, the cells were resuspended in 200–400 μl of PBS and were ready for flow cytometric analysis. All samples were analyzed using Becton Dickinson FACS Calibur flow cytometer (BD Biosciences).

Statistical analysis was performed using SPSS 20.0 software (2000 SPSS for Windows, version 20.0; SPSS Inc., Chicago, Illinois, USA). Analysis of variance (F-test) was used for comparison between three or more groups having quantitative variables. Pearson correlation (r) was used to measure the association between two quantitative variables.


  Results Top


The results showed highly significant difference among the controlled asthma, partly controlled asthma, and uncontrolled asthma groups regarding nocturnal symptoms, limitation of activity, and exacerbation (increased severity of asthma symptoms among groups).

The results showed no significant difference among the controlled asthma, partly controlled asthma, uncontrolled asthma, and control groups regarding liver function tests.

The results showed no significant difference among the controlled asthma, partly controlled asthma, uncontrolled asthma, and control groups regarding kidney function tests.

The results showed highly significant difference among the controlled asthma, partly controlled asthma, uncontrolled asthma, and control groups regarding IgE.

The results showed highly significant difference among the controlled asthma, partly controlled asthma, uncontrolled asthma, and control groups regarding CRP.

The results showed highly significant difference among the controlled asthma, partly controlled asthma, uncontrolled asthma and control groups regarding pulmonary function tests [FEV1% and FEV1/forced vital capacity (FVC%)], as shown in [Figure 1].
Figure 1: Statistical difference among controlled asthma, partly controlled asthma, uncontrolled asthma, and control groups regarding mean pulmonary function tests.

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The results showed highly significant difference among the controlled asthma, partly controlled asthma, uncontrolled asthma, and control groups regarding CD14++ CD16+ cells (P = 0.001), as shown in [Table 1]. The results showed highly significant difference among the controlled asthma, partly controlled asthma, uncontrolled asthma, and control groups regarding mean fluorescence intensity (strength of fluorescence positivity) X (CD16 PE) MX and mean fluorescence intensity (strength of fluorescence positivity) Y (CD14 FITC) MY of CD14++ CD16+ cells as shown in [Table 2]. There is a highly significant correlation between CD14++ CD16+ cells and FEV1 among controlled asthma, partly controlled asthma, uncontrolled asthma, and control groups, as shown in [Figure 2] and [Figure 3].
Table 1: CD14++ CD16+ cells among controlled asthma, partly controlled asthma, uncontrolled asthma and control groups (n=85)

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Table 2: Mean fluorescence intensity of CD14++ CD16+ cells among controlled asthma, partly controlled asthma, uncontrolled asthma, and control groups (n=85)

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Figure 2: Correlation between CD14++ CD16+ cells and forced expiratory volume in 1 s among controlled asthma, partly controlled asthma, uncontrolled asthma, and control group.

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Figure 3: Flow cytometric charts of CD14++ CD16+ (R3) cells. (a) Gating on monocytes. (b) Auto unstained control sample. (c) Stained control sample: the R3 region represents CD14++ CD16 + cells, R3 = 7%. (d) Auto unstained uncontrolled asthmatic patient sample. (e) Stained uncontrolled asthmatic patient sample comparing R3 region between (c) and (e) – confirming increased CD14++ CD16 + cells in asthmatic patients, R3 = 40%. R4: CD14 + CD16 + cells.

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  Discussion Top


Long-term treatment with inhaled corticosteroids, preventive measures, and patient education are the bases of asthma treatment. Bronchodilators such as β2 sympathomimetics are used for rapid symptomatic relief of short-term attacks [9]. 'Global Strategy for Asthma Management and Prevention' named as the Global Initiative for Asthma, where classification of bronchial asthma, is based on the degree of clinical control that can be been achieved, ranging from 'controlled' to 'partly controlled' to 'uncontrolled' asthma [10].

Three monocyte subsets were identified by flow cytometry: CD14++ CD16 − classical monocytes, CD14+ CD16++ nonclassical monocytes, and CD14++ CD16+ intermediate monocytes [11].

This study showed highly significant difference among the controlled asthma, partly controlled asthma, and uncontrolled asthma groups regarding nocturnal symptoms, limitation of activity, and exacerbation, as symptoms of asthma became more worse with increased severity of asthma. This agrees with the results of Mark et al. [12] who reported controlled asthmatic patients should experience no or minimal symptoms, with no limitations in their activities. Moreover, Horan [13] reported that partly controlled asthmatic patients have day symptoms more than twice per week and also showed presence of frequent nocturnal symptoms than controlled patients. There was requirement for medications more than twice per week, and exacerbations also occurred one or more per year, and they had FEV1 less than 80% predicted. Uncontrolled asthmatic patients had three or more features of partly controlled asthma present in any week. This study showed highly significant difference regarding pulmonary function tests among the controlled asthma, partly controlled asthma, uncontrolled asthma, and control groups. FEV1 and FEV1/FVC decreased in partly controlled and uncontrolled asthma group owing to chronic airway inflammation, which can lead to irreversible airway obstruction and deterioration in pulmonary functions whereas these values were normal in controlled asthma group. This agrees with the results of Hong-Ren et al. [14] who reported there was a significant difference in FEV1 and FVC between the controlled and uncontrolled groups as defined the Global Initiative for Asthma guideline. There was also a significant difference in FEV1 and FVC between the partly controlled and uncontrolled groups and the data are presented as mean P value less than 0.05 for the controlled versus uncontrolled group and P value less than 0.05 for the partly controlled versus uncontrolled group.

This study showed highly significant difference among controlled, partly controlled, and uncontrolled asthma groups regarding percentage of CD14++ CD16+ cells, This may be because CD14++ CD16+ monocyte subsets are proinflammatory cells that expressed high levels of C-C motive chemokine receptor 5, and the expression of C-C motive chemokine receptor 5 allowed these cells to migrate to sites of inflammation and produce high levels of proinflammatory cytokines like Tumor necrosis factor-α and IL-1β, which are involved in asthmatic airway inflammation. Therefore, the frequencies of CD14++ CD16+ cells increased significantly in asthma due to inflammatory airway process, leading to increased chronicity and severity of asthma.

This agrees with Moniuszko et al. [15] who reported enhanced frequencies of CD14++ CD16+ monocytes, most significantly in uncontrolled asthmatics as compared with healthy controls and controlled and partly controlled asthmatics, (P < 0.001). Moreover, Yolande et al. [16] reported CD14++ CD16+ monocytes increased in patients with uncontrolled asthma (mean ± SD: 39.5 ± 3.25) compared with partly controlled asthma (mean ± SD: 22.9 ± 2.34) and controlled asthma (mean ± SD: 12.5 ± 1.43). Moreover, this study agrees with Ziegler-Heitbrock and Hofer-Thomas [17] who reported that human peripheral blood CD14++ CD16+ monocyte subsets are the majority of monocytes in severe asthmatics. This study showed highly significant difference among controlled asthma, partly controlled asthma, uncontrolled asthma, and control groups regarding MFIx and MFIy of CD14++ CD16+ cells. The reason of this result is owing to highly increased CD14 density (Mx) with increased chronicity and severity of asthma. Increased CD16 density was an indicator for increased severity of airway inflammation in asthma, and this agree with Tomita et al. [18] who reported the frequencies of CD14++ CD16+ monocytes tended to be increased in controlled, partly controlled, and uncontrolled asthmatics; however, the comparison with healthy participants showed low frequencies. However, this study was in contrast to a study of Skrzeczyńska et al. [8] who reported CD14++ CD16+ subsets in asthmatics are differentially modulated by both the inflammatory process and Glasgow coma score (GCS) treatment; CD14++ CD16+ monocytes had varying responses to GCS treatment, so their frequencies may be affected by GCS treatment in asthmatics.


  Conclusion Top


Human peripheral blood CD14++ CD16+ and CD14+ CD16+ monocyte subsets might be differentially distributed and pharmacologically modulated in asthmatic patients. Most monocytes in uncontrolled asthmatics were of CD14++ CD16+ phenotype.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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Moniuszko M, Bodzenta-Lukaszyk A, Kowal K, Lenczewska D, Dabrowska M. Enhanced frequencies of CD14++CD16+, but not CD14+CD16+, peripheral blood monocytes in severe asthmatic patients. Clin Immunol 2009; 130:338–346  Back to cited text no. 15
    
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2]



 

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