Th17/Th1 phenotype in demyelinating disease

doi:10.1016/j.cyto.2009.12.003

Cytokine

Volume 50, Issue 1, April 2010, Pages 19-23

https://doi.org/10.1016/j.cyto.2009.12.003 Get rights and content

Abstract

Background: Th17 cells are thought to contribute to the immunopathology of allergic and autoimmune conditions. Their role in multiple sclerosis (MS) pathology remains to be fully elucidated. Objective: To assess peripheral blood Th17 responses in patients with MS compared to controls. Methods: We isolated peripheral blood mononuclear cells from 41 MS patients and 23 healthy controls, which were then stimulated using phorbol ester and ionomycin, labelled for CD3, CD8, CD154, IL-17 and IFN-γ and analysed using flow cytometry. Results: Minimal IL-17 was detectable in unstimulated cells. Following stimulation with phorbol ester and ionomycin, PBMCs taken from MS patients in relapse developed a more inflammatory profile than those taken from controls or non-relapse patients, with greater expression of CD154, IL-17 and dual expression of IL-17/IFN-γ. Conclusion: We suggest a greater tendency to Th17 and Th1/Th17 response to non-specific stimulation in MS patients in relapse compared to controls and non-relapse patients.

Introduction

The disordered immunopathology in multiple sclerosis (MS) has long been recognised. One relatively newly-recognised potential contributor to this complex autoimmune condition is the Th17 cell, a markedly pro-inflammatory T-helper cell which, among other factors, secretes IL-17.

T-helper cell polarisation in MS has been implicated as part of the demyelinating disease’s pathology for many years, with initial suggestions that it was a Th1 rather than a Th2 polarised disease, as evidenced by increased levels of Th1 cytokines in MS patients compared to controls, particularly during relapse, with increased Th2 cytokines seen during remission [1], clinical and haematological exacerbations seen following IFN-γ administration [2] and its apparent co-existence with other Th1-type diseases [3], [4] and decreased association with Th2 diseases [5].

However, further work has brought some of these ideas into question, as Th1 (including MS) and Th2 diseases have been shown to be associated with each other [6], [7], and increasing evidence has suggested that the theory of simple Th1/Th2 polarisation may be an oversimplification of complex interactions [8], [9].

The recent discovery of Th17 cells may help to clarify the cytokine profile of MS further. These pro-inflammatory cells have been implicated in the pathology of a variety of allergic and autoimmune conditions, including inflammatory bowel disease [10], psoriasis [11], rheumatoid arthritis [12] and uveitis [13]. There is also an increasing body of evidence that they are involved in MS; the extent of this involvement has yet to be elucidated.

Histopathological work has shown upregulation of IL-17 mRNA in MS lesions [14]. IL-17 mRNA has been shown to be higher in MS CSF and blood compared to controls, with further increase being seen at the time of relapse [15]. In addition, recent work using myelin basic protein-stimulated peripheral blood cells taken from MS patients or controls revealed a subset of MS patients who produced IL-17; levels of which correlated with active lesions on magnetic resonance imaging [16].

Different markers are used to identify the Th17 subset. Essentially, “pan-Th17 cells” are CD4+ T cells, which express or secrete IL-17. A subgroup of these cells also express IFN-γ (denoted Th1/Th17 cells) and/or IL-22 and IL-6 [17], [18]. Other markers which have been investigated include the chemokine receptor, CCR6 [17], [19] and the retinoic acid-related orphan receptor RORC variant 2 which is a key transcription factor for Th17 cell development [10], [17].

The expression of CD154 has not been studied on Th17 cells. CD154 is recognised to be a useful marker for activated T cells. Its expression is short lived and its ligation has multiple (primarily pro-inflammatory) effects on B cells and antigen presenting cells [20], [21], [22]. Its expression has been shown to be elevated in MS compared to controls in peripheral blood [23], [24], [25] and, at autopsy studies, around MS lesions [26].

Based on this evidence, we elected to investigate the IL-17 and IFN-γ response of peripheral blood mononuclear cells (PBMC) taken from patients and controls to non-specific cell activation, using phorbol dibutyrate and ionomycin, and the relationship between CD154 and IL-17 expression in patients with multiple sclerosis and controls.

Section snippets

Materials and methods

Ethical approval was given by the Nottingham Research Ethics Committee 2.

All healthy volunteers and patients gave written informed consent.

Peripheral venous blood was obtained from patients and healthy controls. PBMC were isolated using standard density gradient centrifugation with Histopaque (Sigma), activated using 20 ng/ml phorbol dibutyrate and 0.5 μg/ml ionomycin and incubated at 37 °C in 5% CO₂ for 16 h. After the first hour, 10 μg/ml brefeldin A was added.

Cells were fixed using 4%

Results

PBMCs were isolated from 41 patients with MS and 23 non-MS controls.

Mean ages (with ranges) were 48 years (28–70) in the patient group versus 45 years (30–65) in control group (Mann–Whitney U test, p = 0.2).

The patient group contained 29 females and 12 males; the control group 17 females and 6 males.

Disease subtypes included 26 with RRMS, 7 with SPMS, 3 with PPMS, 3 with PRMS and 2 in transition from RR to SPMS.

EDSS in the patient group ranged from 1.0 to 7.0 (EDSS was not calculated for those

Discussion

The key findings are an increase in T cell IL-17, IL-17-IFN-γ and CD154-IL-17 expression in relapsing MS patients, compared to either healthy controls or non-relapse MS patients.

Although our patient numbers are relatively small, the findings are consistent with recent observations regarding T cell IL-17 and IL-17-IFN-γ expression by Durelli and colleagues [28], and Hedegaard and colleagues show more MS specific findings by using MBP stimulation to increase T cell IL-17 secretion in MS patients

Acknowledgments

L. Edwards is in receipt of a Patrick Berthoud Fellowship.

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Th17/Th1 phenotype in demyelinating disease

Abstract

Introduction

Section snippets

Materials and methods

Results

Discussion

Acknowledgments

J Neuroimmunol

Semin Immunol

Immunol Today

J Neuroimmunol

Exp Neurol

J Neurol Sci

Treatment of multiple sclerosis with gamma interferon: exacerbations associated with activation of the immune system

Neurology

Multiple sclerosis and autoimmune diseases

Aust N Z J Med

Multiple sclerosis and chronic inflammatory diseases. A case–control study

Acta Neurol Scand

Asthma, nasal allergies, and multiple sclerosis

J Allergy Clin Immunol