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Noradrenaline modulates CD4+ T cell priming in rat experimental autoimmune encephalomyelitis: a role for the α1-adrenoceptor

  • Ivan Pilipović
  • Ivana Vujnović
  • Zorica Stojić-Vukanić
  • Raisa Petrović
  • Duško Kosec
  • Mirjana Nacka-Aleksić
  • Nebojša Jasnić
  • Gordana LeposavićEmail author
Original Article
  • 27 Downloads

Abstract

Pharmacological blockade of α1-adrenoceptor is shown to influence development of experimental autoimmune encephalomyelitis (EAE), an IL-17-producing CD4+TCR+ (Th17) cell-mediated disease mimicking multiple sclerosis. Considering significance of CD4+ cell priming for the clinical outcome of EAE, the study examined α1-adrenoceptor-mediated influence of catecholamines, particularly those derived from draining lymph node (dLN) cells (as catecholamine supply from nerve fibers decreases with the initiation of autoimmune diseases) for CD4+ cell priming. The results confirmed diminishing effect of immunization on nerve fiber-derived noradrenaline supply and showed that antigen presenting and CD4+ cells synthesize catecholamines, while antigen presenting cells and only CD4+CD25+Foxp3+ regulatory T cells (Tregs) express α1-adrenoceptor. The analysis of influence of α1-adrenoceptor antagonist prazosin on the myelin basic protein (MBP)-stimulated CD4+ lymphocytes in dLN cell culture showed their diminished proliferation in the presence of prazosin. This was consistent with prazosin enhancing effect on Treg frequency and their Foxp3 expression in these cultures. The latter was associated with upregulation of TGF-β expression. Additionally, prazosin decreased antigen presenting cell activation and affected their cytokine profile by diminishing the frequency of cells that produce Th17 polarizing cytokines (IL-1β and IL-23) and increasing that of IL-10-producing cells. Consistently, the frequency of all IL-17A+ cells and those co-expressing GM-CSF within CD4+ lymphocytes was decreased in prazosin-supplemented MBP-stimulated dLN cell cultures. Collectively, the results indicated that dLN cell-derived catecholamines may influence EAE development by modulating interactions between distinct subtypes of CD4+ T cells and antigen presenting cells through α1-adrenoceptor and consequently CD4+ T cell priming.

Keywords

Noradrenaline α1-Adrenoceptor EAE Tregs Th17 CD4+ lymphocyte proliferation 

Notes

Author contributions

GL, ZSV, IP, and IV designed the study. GL, ZSV, and IV wrote the manuscript. GL, ZSV, IP, IV, RP, DK, MNA, and NJ participated in experiments, data analysis and/or interpretation, and critically reviewed and approved the manuscript.

Funding

This work was supported by the Ministry of Education, Science, and Technological Development of the Republic of Serbia (grant number 175050).

Compliance with ethical standards

All experimental procedures and animal care were performed in accordance with the Directive 2010/63/EU of the European Parliament and of the Council on the protection of animals used for scientific purposes (revising Directive 86/609/EEC) and approved by the Animal Care and Use Committee of the Faculty of Pharmacy (permit number 6/12). The experiments complied with the ARRIVE guidelines for reporting animal research.

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Supplementary material

12026_2019_9082_MOESM1_ESM.tif (347 kb)
Online Resource 1 Arterenol failed to increase CD4+ lymphocyte proliferation in dLN cell cultures from EAE rats in the presence of α1-adrenoceptor blocker. (a) Flow cytometry histogram indicates the gating strategy for proliferating cells (cells in S+G2/M phases of the cell cycle), as determined using 7-AAD (Materials and methods section), within CD4+ lymphocytes (gated as shown in right flow cytometry dot plot), following (left flow cytometry dot plot) the elimination of cellular debris and doublets using pulse processing. (b) Bar graph indicates the frequency of proliferating cells among CD4+ lymphocytes from 72 h dLN cell cultures of female and male rats immunized for EAE (EAE rats) following stimulation with MBP in the absence or in the presence of 10-6 M of arterenol (Art), with or without 10-5 M of prazosin (Pz) (Materials and methods section). The data are shown as mean±SEM (n=6). * p<0.05; ** p<0.01; *** p<0.001; # p<0.05; ### p<0.001. # vs Female. a vs unstimulated; b vs MBP; c vs MBP+Art; d vs MBP+Pz (TIF 347 kb)
12026_2019_9082_Fig10_ESM.png (45 kb)
High Resolution Image (PNG 45 kb)
12026_2019_9082_MOESM3_ESM.tif (371 kb)
Online Resource 2 Prazosin did not influence the proliferation of MACS-separated conventional dLN CD4+CD25- cells. Representative flow cytometry contour plot panel indicates the expression of Ki-67 proliferation marker in MACS-separated conventional male EAE rat dLN CD4+CD25- lymphocytes following 72 h in vitro stimulation with anti-CD3/CD28 mAbs in the absence or in the presence of prazosin, and in the absence of cognate stimuli (Control) (Materials and methods section). Fluorescence minus one (FMO) control incubated with isotype-matched control instead of anti-Ki-67 antibody (-Ki-67 Ab) was used to set the cut-off boundary for Ki-67 expression analysis in MACS-sorted CD4+CD25- cells from cell cultures. Statistical significance of differences was assessed using one-way ANOVA followed by Tukey test for post-hoc comparisons. $$ p<0.01. $ vs Control (TIF 371 kb)
12026_2019_9082_Fig11_ESM.png (75 kb)
High Resolution Image (PNG 75 kb)

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Ivan Pilipović
    • 1
  • Ivana Vujnović
    • 1
  • Zorica Stojić-Vukanić
    • 2
  • Raisa Petrović
    • 1
  • Duško Kosec
    • 1
  • Mirjana Nacka-Aleksić
    • 3
  • Nebojša Jasnić
    • 4
  • Gordana Leposavić
    • 3
    Email author
  1. 1.Immunology Research Centre “Branislav Janković”Institute of Virology, Vaccines and Sera “Torlak”BelgradeSerbia
  2. 2.Department of Microbiology and ImmunologyUniversity of Belgrade-Faculty of PharmacyBelgradeSerbia
  3. 3.Department of PathobiologyUniversity of Belgrade-Faculty of PharmacyBelgradeSerbia
  4. 4.Institute for Physiology and BiochemistryUniversity of Belgrade-Faculty of BiologyBelgradeSerbia

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