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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 30  |  Issue : 3  |  Page : 912-917

Immunomodulatory effect of mesenchymal stem cells on lymphocytes of rheumatoid arthritis patients


1 Department of Clinical Pathology, Faculty of Medicine, Menoufia University, Shebin El-Kom, Menoufia, Egypt
2 Department of Physical Medicine and Rehabilitation, Faculty of Medicine, Menoufia University, Shebin El-Kom, Menoufia, Egypt
3 Department of Clinical Pathology, Shebin El-Kom Teaching Hospital, Shebin El-Kom, Menoufia, Egypt

Date of Submission20-Sep-2016
Date of Acceptance25-Nov-2016
Date of Web Publication15-Nov-2017

Correspondence Address:
Mai F Mohamed Abd El Rahim Badr
Shebin El-Kom, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1110-2098.218291

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  Abstract 

Objectives
The aim of this work was to isolate, expand mesenchymal stem cells (MSCs) from bone marrow (BM), and assess the immunomodulation potential of MSCs on T lymphocytes of patients with rheumatoid arthritis (RA).
Background
MSCs are multipotent adult stem cells present in all tissues. They are present in the BM, and possess remarkable immunomodulation properties that can inhibit the proliferation and function of the major immune cell populations. These unique properties make MSCs ideal candidates for clinical application in autoimmune diseases (e.g., RA).
Patients and methods
MSCs were cultured from BM aspirate and detected morphologically; peripheral blood (PB) mononuclear cells (MNCs) were separated from 30 RA patients and co-culture of BM-MSCs and PB-MNCs were set up. The sample groups were divided into the following groups: group 1, PB MNC culture without phytohemagglutinin (PHA) (negative control); group 2, PB MNC culture with PHA at a concentration of 10 μg/ml; and group 3, PB mononuclear culture with PHA and MSCs. The lymphocytes were harvested and their CD4+ and CD8+ were analyzed by means of flowcytometry.
Results
The results of this study showed a statistically highly significant difference between percentage of CD4+ and CD8+ T lymphocytes cultured with PHA and percentage of CD4+ and CD8+ T lymphocytes cultured without PHA (P < 0.001). There was also a statistically highly significant difference between percentage of CD4+ and CD8+ T lymphocyte cultured without MSCs and percentage of CD4+ and CD8+ T lymphocytes cultured with MSCs, being lower in culture with MSCs (P < 0.001).
Conclusion
MSCs have immunomodulatory effect on T lymphocytes of RA patients.

Keywords: immunomodulation, mesenchymal stem cells, rheumatoid arthritis


How to cite this article:
ELBassuoni MA, Tawfeek GA, Esaily HA, Mohamed Abd El Rahim Badr MF. Immunomodulatory effect of mesenchymal stem cells on lymphocytes of rheumatoid arthritis patients. Menoufia Med J 2017;30:912-7

How to cite this URL:
ELBassuoni MA, Tawfeek GA, Esaily HA, Mohamed Abd El Rahim Badr MF. Immunomodulatory effect of mesenchymal stem cells on lymphocytes of rheumatoid arthritis patients. Menoufia Med J [serial online] 2017 [cited 2019 Nov 17];30:912-7. Available from: http://www.mmj.eg.net/text.asp?2017/30/3/912/218291


  Introduction Top


Rheumatoid arthritis (RA) is a chronic, inflammatory, destructive, and sometimes deforming collagen disease of the joints that has an autoimmune component [1]. Disease-modifying antirheumatic drugs (DMARDs) represent the most important measure in the successful treatment of RA. These agents can retard or prevent disease progression, and thus joint destruction and subsequent loss of function. Moreover, successful DMARD therapy may eliminate the need for other anti-inflammatory or analgesic medications; however, question remains as regards their side effects (e.g., serious infections, malignancies, liver toxicity, renal toxicity, bone marrow (BM) depression, lung inflammation, and skin manifestations [2]. Thus, there is a special need for a new alternative therapy design. Mesenchymal stem cells (MSCs) are multipotent stromal cells that can differentiate into a variety of lineages, including osteocytes, adipocytes, and chondrocytes. This differentiation capacity, in addition to their release of trophic factors and immunomodulatory properties, holds great promise for cell therapies and tissue engineering [3]. MSCs possess remarkable immunosuppressive properties and can inhibit the proliferation and function of the major immune cell populations, including T cells, B cells, and natural killer cells, modulate the activities of dendritic cells, and induce regulatory T cells bothin vivo and in vitro. These unique properties make MSCs ideal candidates for clinical application as immunosuppressants [4], and provide a rational basis for their application in the treatment of immune-mediated diseases (e.g., graft versus host disease, acquired aplastic anemia, multiple sclerosis, RA, Crohn's disease, systemic lupus erythematosis [5]. As regards RA, some studies showed that MSCs are able to decrease arthritis in RA animal models [6]; however, in other studies, the immunosuppressive effect of MSCs might be turned off or even switched to stimulatory effect [7],[8],[9]. Therefore, the role of MSCs in RA remains unclear in many aspects. To clarify this, we studied the immunomodulatory potential of MSCs on the lymphocytes of RA patients.

The aim of this work was to evaluate the immunomodulatory effect of human MSCs on the lymphocytes of RA patients.


  Patients and Methods Top


Reagents

Calcium-free and magnesium-free PBS was purchased from Invitrogen (Faraday Ave, Carlsbad, California, USA). Ficoll-Hypaque solution (density: 1.077 g/l) was purchased from Biochrom AG (Leonorenstr, Berlin). Sterile cell culture low-glucose (1000 mg/l)-Dulbecco's modified Eagle's medium with l-glutamine (2 mmol/l), penicillin/streptomycin, trypsin EDTA 0.25%, fungizone (0.25 μg/ml), and fetal bovine serum were purchased from Lonza (Verviers, Belgium). Sterile cell culture Roswell Park Memorial Institute medium with l-glutamine, phytohaemagglutinin, mitomycin c, and trypan blue 0.4% was purchased from Sigma (Saint Louis, Missouri, USA). Fluorescein isothiocyanate antihuman CD4 and phycoerythrin antihuman CD8 were purchased from Immunostep (Salamanca, Spain).

The present study included 30 patients with RA between 20 and 68 years of age in the Clinical Pathology Department, Menoufiya University Hospital, Menoufiya, Egypt. The diagnosis of RA in patients was based on 2010 revised the American College of Rheumatology and the European League Against Rheumatism classification criteria. Approval of the ethics committee of Menoufia University Hospitals and patient consent were obtained. All RA patients in this study were subjected to the following:

History taking

History taking included name, age, sex, and complaint taken in the patient's own words, emphasizing on the disease duration and pain with the evaluation of its severity.

Clinical examination

Laboratory investigations: C-reactive protein, erythrocyte sedimentation rate, rheumatoid factor, and anticyclic citrullinated peptide were evaluated.

In this study, 10 ml was aspirated from normocellular BM donors under aseptic condition. Mononuclear cells (MNCs) were separated by means of density gradient centrifugation and cultured in 25 cm2 flasks for isolation, purification, and expansion of MSCs.

Peripheral blood (PB) of 5 ml was obtained from RA patients, the tubes were centrifuged, and the MNC fraction was collected. The MSCs were co-cultured with MNCs of RA patients at a ratio of MSCs:MNCs 1:4.

The sample groups were divided as follows: group 1, PB MNC culture without PHA (negative control); group 2, PB MNC culture with PHA; and group 3, PB MNC with PHA and mesenchymal stem cells.

After 72 h of the culture, the cells were harvested and analyzed by means of flowcytometry.

Statistical analysis

The data collected were tabulated and analyzed using Statistical Package for Social Science, version 22.0 (Illinois, Chicago, USA) on IBM compatible computer.

Two types of statistics were performed: descriptive statistics (e.g., percentage (%), mean, and SD), and analytic statistics (paired t-test is a test of significance used for comparison between two related groups having normally distributed quantitative variables). The Wilcoxon signed-rank test (nonparametric test) is a test of significance used for comparison between two related groups having not normally distributed quantitative variables. Pearson correlation (r) is a test used to measure the association between two normally distributed quantitative variables. Spearman's correlation coefficient (r) (nonparametric test) is a test used to measure the association between two not normally distributed quantitative variables. Level of significance was set as P-value less than 0.05.


  Results Top


The table shows that there were 30 RA patients. Their ages ranged between 20 and 68 years (X̄±SD = 45.0±11.8). As regards sex, 3.3% were male (one case) and 96.7% were female (29 cases) [Table 1] and [Figure 1].
Table 1: Demographic characteristics of rheumatoid arthritis patients

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Figure 1: Demographic characteristics of rheumatoid arthritis patients.

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The table shows that the duration of disease ranged between 2 and 7 years (3.9 ± 1.3), the number of swollen joint ranged between 0 and 10 (2.6 ± 2.4), and the number of tender joint ranged between 0 and 26) (5.9 ± 6.3) [Table 2] and [Figure 2].
Table 2: Clinical characteristics of rheumatoid arthritis patients

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Figure 2: Clinical characteristics of rheumatoid arthritis patients.

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The table shows that erythrocyte sedimentation rate value ranged from 15 to 85 (X̄±SD = 46.6±20.3), C-reactive protein value ranged from 2 to 72 (X̄±SD = 20.9±19.4), rheumatoid factor value ranged from 5 to 128 (X̄±SD = 34.9±35.0) and anticyclic citrullinated peptide value ranged from 2 to 267 (X̄±SD = 67.6±71.6) [Table 3] and [Figure 3].
Table 3: Routine investigations of rheumatoid arthritis patients

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Figure 3: Routine investigations of rheumatoid arthritis patients.

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There was a highly significant difference between the percentage of CD4+ and CD8+ T lymphocytes cultured without PHA and CD4+ and CD8+ T lymphocytes cultured with PHA, being higher in culture with PHA [Table 4] and [Figure 4].
Table 4: Effect of phytohemagglutinin on cultured lymphocytes

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Figure 4: Effect of phytohemagglutinin on cultured lymphocytes.

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There was a highly significant difference between the percentage of CD4+ and CD8+ T lymphocytes cultured without MSCs and CD4+ and CD8+ T lymphocytes cultured with MSCs, being lower in culture with MSCs [Table 5] and [Figure 5], [Figure 6], [Figure 7].
Table 5: Effect of mesenchymal stem cells on cultured T lymphocytes of patients with rheumatoid arthritis

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Figure 5: Bone marrow-derived mesenchymal stem cells (MSCs) showed fibroblastoid adherent cells with 80% confluence (a). MSCs cocultred with peripheral blood mononuclear cells (b).

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Figure 6: Effect of mesenchymal stem cells on cultured T lymphocytes of patients with rheumatoid arthritis.

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Figure 7: Flowcytometric analysis of CD4 and CD8 expression of lymphocytes without mesenchymal stem cells (MSCs) (a) and with MSCs (b).

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  Discussion Top


MSCs are largely studied as modulators of immune responses in a variety of diseases related to alloreactive immunity or autoimmunity [10]. RA is a systemic autoimmune disease and presents an inflammatory environment with immunological involvement, and this has been an obstacle that can potentially limit the use of cartilage tissue engineering [11]. MSCs have generated great interest for their ability to display immunomodulatory capacities bothin vitro and in vivo, as well as in the alleviation of joint inflammation and destruction [12], and thus they hold great potential as a new alternative effective tool for treating many immune-mediated diseases such as RA. In the light of the previous postulations, the aim of this study was to investigate the immunomodulatory effect of MSCs on lymphocytes of RA patients.

In the current study, there was a significant increase in CD4+ and CD8+ T lymphocytes cultured with PHA than in CD4+ and CD8+ T lymphocytes cultured without PHA. This is in agreement with the findings of Lim et al.[13], who demonstrated the efficacy of measuring T-lymphocyte activation as a measure of T-lymphocyte function by determining the proportion of activated CD69 T lymphocytes using flowcytometry. They found that the concentration of 20 μg of PHA per ml was optimal in stimulating the largest proportion of CD4+ and CD8+ T lymphocytes. However, Duarte et al. [14] showed that the expansion of T cells with PHA impairs their ability to respond (proliferation, cytotoxicity, and IFN-γ and perforin expression) to allogeneic stimulation or viral antigens in vitro.

There was a highly significant difference between the percentage of CD4+ and CD8+ T lymphocytes cultured without MSCs and CD4+ and CD8+ T lymphocytes cultured with MSCs, being lower in cultures with MSCs. This is in agreement with the findings ofCarrión et al. [15], who showed that early addition (day 0) of MSCs suppressed all CD4+ and CD8+ T cell lineages (highly significant difference) (P < 0.0001). However, Djouad et al. [16] observed that, in the collagen-induced arthritis model of arthritis, MSCs did not confer any benefit. Both the clinical and the immunologic findings suggested that MSCs were associated with accentuation of the Th1 response, and they suggested that environmental parameters, in particular those related to inflammation, might influence the immunosuppressive properties of MSCs.


  Conclusion Top


The present study proved the immunomodulatory effect of MSCs on lymphocytes of RA patients, as the co-culture of BM mesenchymal stem cells with MNCs of RA patients shows a clinically significant statistical decrease in the percentage of CD4+ T lymphocytes and CD8+ T lymphocytes.

We hope that BM-derived MSCs will play a more crucial role in the future therapy of RA patients and translate its therapeutic potential into real clinical application.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
  References Top

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Venables PJW, Ravinder NM. Diagnosis and differential diagnosis of rheumatoid arthritis. Topic J 2015; 13:7504–7550.  Back to cited text no. 1
    
2.
Pawade RB, Bhalerao PP, Kunkulol RR, Kute NS. Disease-Modifying Anti-Rheumatic Drugs (DMARDs) used for rheumatoid arthritis – a review. Indian J Basic Appl Med Res 2015; 4:272–288.  Back to cited text no. 2
    
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Murray IR, Péault B. Q & A: mesenchymal stem cells – where do they come from and is it important? BMC Biol2015; 13:99.  Back to cited text no. 3
    
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SullivanC, Murphy JM, Griffin MD, Porter RM, Evans CH, O'Flatharta C, et al. Genetic mismatch affects the immunosuppressive properties of mesenchymal stem cells in vitro and their ability to influence the course of collagen-induced arthritis. Arthritis Res Ther 2012; 14:167.  Back to cited text no. 7
    
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Papadopoulou A, Yiangou M, Athanasiou E, Zogas N, Kaloyannidis P, Batsis I, et al. Mesenchymal stem cells are conditionally therapeutic in preclinical models of rheumatoid arthritis. Ann Rheum Dis 2012; 71:1733–1740.  Back to cited text no. 8
    
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Schurgers E, Kelchtermans H, Mitera T, Geboes L, Matthys P. Discrepancy between the in vitro and in vivo effects of murine mesenchymal stem cells on T-cell proliferation and collagen-induced arthritis. Arthritis Res Ther 2010; 12:31.  Back to cited text no. 9
    
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Ghannam S, Bouffi C, Djouad F, Jorgensen C, Noël D. Immunosuppression by mesenchymal stem cells: mechanisms and clinical applications. Stem Cell Res Ther 2010; 1:2.  Back to cited text no. 10
    
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McInnes IB, Schett G. The pathogenesis of rheumatoid arthritis. N Engl J Med 2011; 365:2205–2219.  Back to cited text no. 11
    
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Mattar P, Bieback K. Comparing the immunomodulatory properties of bone marrow, adipose tissue, and birth-associated tissue mesenchymal stromal cells. Front Immunol 2015; 3:560.  Back to cited text no. 12
    
13.
Lim LCL, Fiordalisi MN, Mantell JL, Schmitz JL, Folds JD. A whole-blood assay for qualitative and semiquantitative measurements of CD69 surface expression on CD4 and CD8 T lymphocytes using flow cytometry. Clin Diagn Lab Immunol 1998; 5:392–398.  Back to cited text no. 13
    
14.
Duarte RF, Chen FE, Lowdell MW, Potter MN, Lamana ML, Prentice HG, et al. Functional impairment of human T-lymphocytes following PHA-induced expansion and retroviral transduction: implications for gene therapy. Gene Ther 2002;9:1359–1368.  Back to cited text no. 14
    
15.
Carrión F, Nova E, Luz P, Apablaza F, Figueroa F. Opposing effect of mesenchymal stem cells on Th1 and Th17 cell polarization according to the state of CD4+T cell activation. Immunol Lett 2011; 135:10–16.  Back to cited text no. 15
    
16.
Djouad F, Fritz V, Apparailly F, Louis-Plence P, Bony C, Sany J, et al. Reversal of the immunosuppressive properties of mesenchymal stem cells by tumor necrosis factor alpha in collagen-induced arthritis. Arthritis Rheum 2005; 52:1595–1603.  Back to cited text no. 16
    


    Figures

  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4], [Table 5]



 

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