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 Table of Contents  
ORIGINAL ARTICLE
Year : 2016  |  Volume : 33  |  Issue : 1  |  Page : 14-18

Expression of intracellular interleukin-17 in systemic lupus erythematosus patients


1 Department of Clinical and Chemical Pathology, Faculty of Medicine, Benha University, Benha, Egypt
2 Department of Rheumatology and Rehabilitation, Benha Teaching Hospital – General Organization of Teaching Hospitals and Institutes, Benha, Egypt

Date of Submission04-May-2016
Date of Acceptance09-May-2016
Date of Web Publication28-Nov-2016

Correspondence Address:
Seham G Ameen
Department of Clinical and Chemical Pathology, Faculty of Medicine, Benha University, Banha
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-208X.194382

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  Abstract 


Background Many significant evidences implicate interleukin-17 (IL-17) in inflammation, autoimmunity, and defense against some bacteria. One of the chronic autoimmune diseases in which IL-17 might be involved in is systemic lupus erythematosus (SLE). Evidences have identified IL-17 as a key player in the pathogenesis of SLE.
Aim of the study The aim of this study was to verify how IL-17 was involved in the immune response in patients with SLE.
Patients and methods Forty diagnosed cases of SLE and 20 apparently healthy individuals serving as a control group were recruited for this case–control study from Benha University Hospital and Benha Teaching Hospital. Sterile peripheral blood samples were collected from the study group and the percentage of IL-17 expressing T-cell subpopulation was determined by using flow cytometry.
Results After suitable statistical analysis, our results showed a high percentage of intracellular IL-17 in SLE patients and a high statistical increase in the intensity of IL-17 in CD3+, CD4+, and CD8+ T cells in SLE cases when compared with the control group.
Conclusion High percentage and intensity of IL-17 in T-cell subpopulation reflect its close involvement in the inflammatory immune response, which provides an evidence of its role in the pathogenesis of SLE.

Keywords: flow cytometry, interleukin-17, systemic lupus erythematosus


How to cite this article:
Abd El-Gwad ER, Elshabacy FA, Abdul-Hafeez NA, Ameen SG. Expression of intracellular interleukin-17 in systemic lupus erythematosus patients. Benha Med J 2016;33:14-8

How to cite this URL:
Abd El-Gwad ER, Elshabacy FA, Abdul-Hafeez NA, Ameen SG. Expression of intracellular interleukin-17 in systemic lupus erythematosus patients. Benha Med J [serial online] 2016 [cited 2020 Jul 7];33:14-8. Available from: http://www.bmfj.eg.net/text.asp?2016/33/1/14/194382




  Introduction Top


Systemic lupus erythematosus (SLE) is a prototypical systemic autoimmune disease in which tolerance against ubiquitous self-antigens is lost [1]. The profound heterogeneity of SLE causes problems regarding diagnostic accuracy in clinical practice and particularly in clinical research [2].

Clinical symptoms are heterogeneous and range from mild skin rashes to more severe multiorgan manifestations, primarily involving the kidney, the brain, and blood [3]. The clinical diagnosis of SLE relies on the identification of markers of systemic tolerance failure, for example, antinuclear and antidouble stranded DNA antibodies, and is associated with the presence of target organ involvement, for example, glomerulonephritis and skin rash [4]. Mechanisms of tissue damage in patients with SLE involve autoantibody and immune complex deposition, as well as infiltration of tissues by lymphocytes [5].

This condition is characterized by both innate and adaptive immune dysregulation but the relative importance of cytokines operative in these systems is questionable [6]. The disease course is unpredictable, with episodes of flares and remissions, sometimes leading to permanent organ damage and preterm death [2].

Chronic immune activation in SLE leads to the production of large amounts of inflammatory cytokines and contributes actively to local inflammation and tissue damage [7].

These cytokines may exert either proinflammatory or anti-inflammatory effects, or both, depending on the specific local microenvironment, thus contributing greatly to SLE pathogenesis. Understanding these cytokine abnormalities may be beneficial in developing effective targeting therapy [8].

Among those cytokines is interleukin-17 (IL-17), which is produced by several cell subsets including CD4+ T cells, CD8+ T cells, γδ T cells, natural killer cells, neutrophils, dendritic cells, microglia, eosinophil, astrocytes, and oligodendrocytes [9].

IL-17 has been associated with the pathogenesis of a range of autoimmune diseases, including rheumatoid arthritis, systemic sclerosis, multiple sclerosis, and SLE [6]. It promotes inflammation by inducing local chemokine and cytokine secretion and is essential for the clearance of certain pathogens such as bacteria and fungi. Moreover, IL-17 has been linked to the instigation of immune-mediated organ damage in the context of several autoimmune diseases [8].

Multiple lines of evidence have identified IL-17 as a key player in the pathogenesis of SLE. Lupus mice deficient in IL-17 or IL-17 receptors were shown to be protected from lupus nephritis [10].

Diverse cytokine abnormalities common in patients with SLE may skew the differentiation of T cells into IL-17-producing CD4+ and double negative T cells. This phenomenon could promote the autoimmune process by increasing the activation of immune cells and stimulating B-cell proliferation and antibody production [4].

The objective of this study was to evaluate whether IL-17 is involved in autoimmune response in patients with SLE, and to assess the proportion of intracellular IL-17 in a T-cell subpopulation.


  Patients and methods Top


Patients

After the case-controlled study design was approved by the scientific research and ethics committee at our organization, 60 individuals were selected according to the inclusion and exclusion criteria. They comprised 40 SLE patients fulfilling the criteria of the American College of Rheumatology [11] (37 women and three males diagnosed with SLE) with their ages ranging between 18 and 48 years (mean±SD; 31±8.7). They were recruited from the outpatient clinic at our place.

In addition, 20 apparently healthy individuals served as the control group (18 women and two men). Their ages ranged between 18 and 40 years (36.3±2.13). They were age and sex-matched with the patients in the SLE group.

Sterile blood samples were collected from each of the participants in our study and the following investigations were carried out: complete blood count, erythrocyte sedimentation rate, serum creatinine, 24 h protein in urine (for SLE cases only), anti-DNA, antinuclear antibodies levels, and the flow cytometric analysis for IL-17 expression and intensity in CD3+, CD4+, and CD8+ T cells.

Methods

Flow cytometry for IL-17 expression and intensity in CD3+, CD4+, and CD8+ T cells was carried out using the following.

Antibodies and reagents

Cell surface staining anti-CD3-fluorescein isothiocyanate, anti-CD4-fluorescein isothiocyanate, anti-CD8-Per-CP, and anti-CD45-Per-CP were purchased from BD (San Jose, California, USA). Staining for intracellular cytokines was performed with anti-IL-17-PE purchased from R&D (Minneapolis, Minnesota, USA). The cytofix/cytoperm reagent was purchased from BD.

Flow cytometric analysis

Intracellular staining was performed with the BD cytofix/cytoperm reagent according to the manufacturer’s instructions. For the analysis of the cell population and cytokine production, a first gate that included live lymphocytes (according to forward scatter and side scatter characteristics) was used. Next, the expression of CD3 and IL-17 on this population was assessed (CD3 vs. IL-17) according to this gate. Then the cells were identified by assessing the expression of CD4, CD8, and IL-17.

For data analysis, stained cells were acquired on a fluorescence-activated cell sorting caliber flow cytometer (BD Biosciences, Franklin lakes, New Jersey, USA), which was set up using validated quality assurance procedures. Analysis was performed with the CellQuest software program (Mac OS9, version 5.1, San Diego, California, USA).

Statistical analysis

Excel program (Microsoft Office 2013) and statistical package for social science program, version 20 were used for statistical analyses (SPSS Inc., Chicago, Illinois, USA). Results were expressed as mean, SD, median, and range. Correlation coefficients were used to correlate numeric variables (Pearson’s correlation for parametric and Spearman’s correlation for nonparametric). IL-17 intensity was calculated by using the percentage of cells containing intracellular IL-17 divided on the percentage of the same cells present in the peripheral blood.


  Results Top


The most frequent clinical variables among SLE patients at the time of taking samples were arthritis (72.5%), malar rash (70%), alopecia (62.5%), and oral ulcers (62.5%).

The routine laboratory investigations showed a highly statistically significant decrease in hemoglobin concentration and a highly statistically significant increase in erythrocyte sedimentation rate in the SLE group when compared with the control group.

All SLE patients had positive antinuclear antibodies; 95% were antidouble stranded DNA positive and 75% had proteinuria.

Frequency and cellular source of interleukin-17

The fraction of IL-17 expressing T cells was higher in SLE patients than in controls; however, this did not reach statistical significance (mean=92.7 vs. 90.5, P=0.594).

To investigate which T-cell subpopulation was responsible for the production of IL-17, we performed multicolor flow cytometry experiments.

The production of IL-17 was detected in a small proportion of CD3+ T cells; however, it was higher in SLE patients than in controls (57 vs. 53, P=0.210).

The intensity of IL-17 in CD3+ T cells was highly significantly increased in SLE patients than in controls (94.6 vs. 81.2, P<0.001).

Receiver operating characteristic (ROC) curves for the percentage of CD3+, CD3+IL-17+, and IL-17 intensity in CD3+ T cells were plotted for comparison between SLE cases and healthy controls. IL-17 intensity in CD3+ showed excellent area under the curve (AUC) for comparison between SLE and controls, and had significantly higher AUC when compared with CD3+ and CD3+IL-17+ (P<0.001 and P=0.001, respectively) ([Figure 1]).
Figure 1 (a) ROC curve for CD3+, CD3+IL-17+, and IL-17 intensity in CD3+ T cells for comparison between SLE and healthy controls. (b) ROC curve for CD4+, CD4+IL-17+, and IL-17 intensity in CD4+ T cells for comparison between SLE and healthy controls. (c) ROC curve for CD8+, CD8+IL-17+, and IL-17 intensity in CD8+ T cells for comparison between SLE and healthy controls. IL, interleukin; ROC, receiver operating characteristic.

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The proportion of CD4+IL-17+ T cells was higher in patients with SLE than in controls; however, it was not statistically significant (35.16 vs. 31.14, P=0.207).

The intensity of IL-17 in CD4+ T cells was highly significantly increased in SLE patients than in controls (96.9 vs. 79, P<0.001).

ROC curves for the percentage of CD4+, CD4+IL-17+, and IL-17 intensity in CD4+ T cells were plotted for comparison between SLE cases and healthy controls. IL-17 intensity in CD4+ showed excellent AUC for comparison between SLE and controls and had significantly higher AUC when compared with CD4+ and CD4+IL-17+ (P<0.001 and P=0.001, respectively).

The SLE patients had a high statistically significant proportion of CD8+IL-17+ T cells than did the healthy controls (23.4 vs. 15.4; P=0.003).

The intensity of IL-17 in CD8+ T cells was highly significantly increased in SLE patients than in controls (73.9 vs. 50.6, P<0.001).

ROC curves for the percentage of CD8+, CD8+IL-17+, and IL-17 intensity in CD8+ T cells were plotted for comparison between SLE cases and healthy controls. IL-17 intensity in CD8+ showed excellent AUC for comparison between SLE and controls and had significantly higher AUC when compared with CD8+ and CD8+IL-17+ (P<0.001, P=0.001, respectively) ([Table 1] and [Table 2]).
Table 1 Flow cytometric values for all studied groups

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Table 2 Intensity of IL-17 in the studied groups

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  Discussion Top


In SLE there is persistent activation of the immune system with production of cytokines [12]. The role of IL-17 in the pathogenesis of SLE is accepted, and is expressed by many types of T cells [6]. Therefore, in the present study, we assessed the proportion of intracellular IL-17 in a T-cell subpopulation by studying the flow cytometric intracellular IL-17 expression in some T-cell subpopulation.

A higher percent of intracellular IL-17 was observed in peripheral blood cells in SLE patients than in controls. However, it has been reported that the proportion of IL-17-producing cells was not significantly increased in SLE patients when compared with healthy controls [13].

We found that the proportion of CD3+IL-17+ T cells was relatively small in SLE cases but it was higher than that in controls. In addition, it has been reported that the proportion of IL-17 expression in CD3+ cells tends to be higher in SLE patients [13].

In the present study, the intensity of IL-17 in CD3+ T cells was significantly higher in SLE patients than in controls. The sensitivity and specificity of intensity of IL-17 in CD3+ T cells was determined for comparison between SLE patients and controls using ROC curves, and we observed that the sensitivity and specificity were 87.5 and 95%, respectively, with a cutoff value of 90.2.

By investigating the proportion of CD4+IL-17+ T cells in SLE cases and controls, the proportion of CD4+IL-17+ T cells was higher in patients with SLE than in controls. However, it was reported that the percentage of CD4+IL-17+ cells was not different between controls and SLE patients; in fact, some controls had a higher percentage of CD4+IL-17+ cells than in SLE patients [14].

In the present study, the intensity of IL-17 in CD4+ T cells was significantly higher in SLE patients than in controls. The sensitivity and specificity of intensity of IL-17 in CD4+ T cells were determined for comparison between SLE patients and controls using ROC curves, and we observed that the sensitivity and specificity were 100 and 100%, respectively, with a cutoff value of 89.7.

CD8+IL-17+ T cells were also evaluated in SLE cases and controls and had a high statistically significant proportion in SLE cases than in controls. However, it has been reported that the percentage of IL-17+ CD8+ cells were not different between controls and SLE patients [14]. Moreover, it has been reported that CD8+ T cells produced minimal amounts of IL-17 compared with CD4+cells [5].

In the present study, the intensity of IL-17 in CD8+ T cells was significantly higher in SLE patients than in controls. The sensitivity and specificity of intensity of IL-17 in CD8+ T cells were determined for comparison between SLE patients and controls using ROC curves and we observed that the sensitivity and specificity were 92.5 and 95%, respectively, with a cutoff value of 62.7.


  Conclusion Top


High percentage and intensity of IL-17 in a T-cell subpopulation reflect its close involvement in inflammatory immune response, which provides an evidence of its role in the pathogenesis of SLE.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Crispín JC, Vasileios C, Kyttaris N, George C, Tsokos GC. The role of interleukin-17 in systemic lupus erythematosus. New York: Springer Science+Business Media; 2011: 391–400  Back to cited text no. 4
    
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Crispín JC, Oukka M, Bayliss G, Cohen RA, van Beek CA, Stillman IE et al. Expanded double negative T cells in patients with systemic lupus erythematosus produce IL-17 and infiltrate the kidneys. J Immunol 2008; 181:8761–8766.  Back to cited text no. 5
    
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Su DL, Lu ZM, Shen MN, Li X, Sun LY. Roles of pro- and anti-inflammatory cytokines in the pathogenesis of SLE. J Biomed Biotechnol 2012; 2012:347141.  Back to cited text no. 8
    
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Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997; 40:1725.  Back to cited text no. 11
    
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Liu MF, Wang CR. Increased Th17 cells in flow cytometer-sorted CD45RO-positive memory CD4 T cells from patients with systemic lupus erythematosus. Lupus Sci Med 2015; 10:1136–1140.  Back to cited text no. 12
    
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Puwipirom H, Hirankarn N, Sodsai P, Avihingsanon Y, Wongpiyabovorn J, Palaga T. Increased IL23 receptor(+) T cells in peripheral blood mononuclear cells of patients with systemic lupus erythematosus. Arthritis Res Ther 2010; 12:1186–1192.  Back to cited text no. 14
    


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    Tables

  [Table 1], [Table 2]



 

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