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Keywords:

  • cellular immunity;
  • cytokines;
  • interferon-beta;
  • multiple sclerosis;
  • treatment response

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

We investigated cellular immune responses at baseline in peripheral blood mononuclear cells (PBMC) of patients with multiple sclerosis (MS) treated with interferon (IFN)-β and classified into responders and non-responders according to clinical response criteria. Levels for IFN-γ, interleukin (IL)-17A, IL-17F, IL-10 and IL-4 were determined in activated PBMC of 10 responders, 10 non-responders and 10 healthy controls by cytometric bead arrays. Cytokine levels in cell culture supernatants were similar between responders and non-responders, and comparable to those obtained in healthy controls. These findings do not support differential cellular immune responses in PBMC at baseline between IFN-β responders and non-responders.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Interferon (IFN)-β has demonstrated beneficial effects in patients with relapsing–remitting multiple sclerosis (RRMS), decreasing the relapse rate and reducing brain disease activity as assessed by magnetic resonance imaging [1-3]. However, the drug is only partially effective, and a relatively large proportion of patients do not respond to IFN-β [4].

In a previous study, we showed that peripheral blood mononuclear cells (PBMC) from IFN-β non-responders were characterized by a baseline over-expression of genes induced by type I IFNs compared to treatment responders [5]. IFN-β belongs to the type I IFN family, which is composed of pleiotropic cytokines of the innate immune system with the ability to modulate adaptive immune responses. In this context, type I IFNs can redirect CD4+ T cells into T helper type I cells (Th1) [6].

In a recent study, using the animal model of the disease, experimental autoimmune encephalomyelitis (EAE) [7], the authors reported that IFN-β blocked cell differentiation to the Th17 phenotype by inducing IFN-γ. They observed that IFN-β was effective in ameliorating EAE symptoms induced by Th1 cells but worsened the disease induced by Th17 cells. The authors also identified a subgroup of IFN-β non-responders characterized by high baseline serum levels of interleukin (IL)-17F [7].

Based on these observations, in the present study we aimed to investigate the type of cellular immune responses occurring at baseline in IFN-β non-responders by determining the cytokine profile of activated PBMC from RRMS patients treated with IFN-β and classified into responders and non-responders according to their clinical response to treatment.

Materials and methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Patients and definition of response to IFN-β therapy

All subjects included in the study satisfied Poser's criteria for clinically definite MS [8]. The study was approved by the local ethics committees and samples were collected with written informed consent.

Clinical criteria for response to IFN-β were applied after 2 years of treatment. Patients were labelled as non-responders if they experienced one or more relapses and an increase of at least 1 point in the Expanded Disability Status Scale (EDSS) score that persisted for a minimum of two consecutive visits separated by a 6-month interval. Patients were classified as responders if they were free of relapses and showed no increase in the EDSS score during the 2-year follow-up period [9]. Twenty RRMS patients, 10 responders and 10 non-responders, and a group of 10 healthy controls were included into the study.

None of these patients had ever received treatment with IFN-β or other immunosuppressive therapy before study entry. No patient had clinical exacerbations or received corticosteroid treatment during the month before initiation of IFN-β. As the analysis of cellular immune responses was focused only on blood samples that were collected before IFN-β treatment, determination of neutralizing antibodies was not considered for the present study.

A summary of the main demographic and baseline clinical characteristics of patients and controls is shown in Table 1.

Table 1. Demographic and baseline clinical and radiological characteristics of multiple sclerosis (MS) patients and controls included in the study
Baseline characteristicsRNRHCP-values§
  1. Data are expressed as mean (standard deviation) unless stated otherwise. *Data are expressed as mean (interquartile range). Refers to the number of relapses in the 2 previous years. Magnetic resonance imaging (MRI) data were available for 12 patients, nine responders and three non-responders. §Refers to P-values obtained following comparisons between interferon (IFN)-β responders and non-responders by means of Student's t-test (age, EDSS, T2LL), Mann–Whitney U-test [disease duration, number of relapses, number of gadolinium (Gd)-enhancing lesions] and χ2 test (type of IFN-β). EDSS: Expanded Disability Status Scale; T2LL: T2 lesion load expressed as percentage of total brain content; i.m.: intramuscular; s.c.: subcutaneous; R: responders to IFN-β; IR: intermediate responders to IFN-β; NR: non-responders to IFN-β; HC: healthy controls.

n101010
Age (years)29·0 (5·9)34·1 (8·9)28·9 (7·2)0·152
Female/male (% women)10/– (100)10/– (100)10/– (100)
Duration of disease (years)1·7 (1·2)5·3 (4·9)0·052
EDSS*1·6 (1·5–2·0)2·3 (2·0–3·0)0·061
Number of relapses1·7 (0·9)2·2 (0·8)0·280
Number of Gd+ lesions3·6 (5·4)0·3 (0·6)0·282
T2LL0·5 (0·4)0·6 (0·8)0·684
Type of IFN-β [n (%)]    
IFN-β1a i.m.4 (40)4 (40) 
IFN-β1b s.c.1 (10)3 (20)0·472
IFN-β1a s.c.5 (50)3 (40) 

Sample collection and cell culture

Peripheral blood was collected from healthy controls and RRMS patients before initiation of treatment with IFN-β. PBMC were isolated by Ficoll-Isopaque density gradient centrifugation (Gibco BRL, Life Technologies Ltd, Paisley, UK) and stored in liquid nitrogen until used. Two × 106 cells were cultured in complete media in the absence or presence of phorbol 12-myristate 13-acetate (PMA) plus ionomycin calcium salt (IO) (both from Sigma Chemical Co., St Louis, MO, USA) at 50 ng/ml and 1 μg/ml concentrations, respectively. After 24 h incubation at 37°C and 5% CO2, cells were centrifuged and supernatants collected and stored at −80°C until used.

Determination of levels for IFN-γ, IL-17A, IL-17F, IL-10 and IL-4 in cell culture supernatants

Cytokine levels were determined in cell supernatants using the cytometric bead array system (CBA) (Bender MedSystems®, San Diego, CA, USA). A 4-plex assay was performed for IFN-γ, IL-17A, IL-10 and IL-4, and a simplex assay was carried out for IL-17F detection. The procedure was performed following the manufacturer's instructions. Beads were acquired using a dual-laser fluorescence activated cell sorter (FACS)Canto (Becton Dickinson, Mountain View, CA, USA) and analysed using FlowCytomix Pro Software.

Statistical analysis

Parametric analysis of the variance was performed, after checking the normality of the variables, to compare group effect with cytokine levels, adjusting for between-experiments batch effects. Statistical calculations were performed using the R program.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

PBMC obtained at baseline from 20 RRMS patients, 10 responders and 10 non-responders, were activated with a combination of PMA and IO. After 24 h, levels of IFN-γ, IL-10, IL-4, IL-17A and IL-17F were determined in cell culture supernatants by means of CBAs. As shown in Fig. 1, cytokine levels were similar between responders and non-responders, and none of the comparisons between groups revealed statistically significant differences (P > 0·05). Similarly, IFN-γ, IL-10, IL-4, IL-17A and IL-17F levels in responders and non-responders were comparable to the cytokine levels observed in a healthy control group of 10 individuals whose PBMC were cultured in similar conditions (P > 0·05 for all comparisons) (Fig. 1).

figure

Figure 1. Baseline cellular immune responses in peripheral blood mononuclear cells (PBMC) of responders, non-responders and healthy controls. Graph bars showing levels of interferon (IFN)-γ, interleukin (IL)-4, IL-17F, IL-17A and IL-10 obtained in cell culture supernatants from activated PBMC of patients with relapsing–remitting multiple sclerosis (RRMS) classified by their clinical response to IFN-β treatment. Cytokine levels were compared with a control group. Graphs represent mean cytokine levels and standard error of the mean (s.e.m.) (165 × 111 mm; 300 × 300 DPI).

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

Type I IFNs are known to favour Th1-type immune responses [6]. Th1 responses are activated mainly for battling viral infections and IFN-β, a type I IFN, has a potent effect in controlling viral invasion [10]. In addition, IFN-β has been shown to increase CD8+ T cell immune responses and other mechanisms to manage a viral infection [11]. Recently, several studies have suggested a potential link between response to IFN-β in MS patients and particular types of cellular immune responses. In a previous study [5], we showed that PBMC from non-responders to IFN-β were characterized by a baseline type I IFN signature compared to IFN-β responders, which suggested that the over-expression of type I IFN responsive genes could indirectly induce immune responses of Th1 type in non-responders.

In a study by Axtell et al. [7], the authors associated a poor response to IFN-β treatment with Th17-type immune responses in EAE mice. Supporting the EAE data, the authors identified elevated pretreatment serum levels of IL-17F in a small subgroup of IFN-β non-responders. Along the same lines, Lee et al. [12] reported positive correlations between high serum levels of IL-7 in RRMS patients and a good response to IFN-β treatment, and in-vitro experiments revealed Th1 differentiation induced by IL-7. However, these findings were not validated in a recent study [13].

In this study, we aimed to investigate the type of immune responses (Th1, Th2, Th17) present in PBMC obtained at baseline from RRMS patients and classified based on their clinical response to IFN-β treatment. For this, levels of IFN-γ, IL-10, IL-4, IL-17A and IL-17F were determined in culture supernatants from activated PBMC of responders and non-responders and also from healthy controls. Cytokine levels were similar between groups. Although these results are based on a relatively small number of responders and non-responders to IFN-β, the findings do not support an association between differential responses to IFN-β and Th1, Th2 or Th17 types of immune responses. However, it should be taken into account that stimulation with PMA plus IO is associated with a strong and general PBMC activation, and therefore it remains unknown whether the use of more specific T cell activation, such as that provided by CD3 stimulation, may result in significant differences of the cellular immune responses between IFN-β responders and non-responders.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References

The authors thank the Red Española de Esclerosis Múltiple (REEM) sponsored by the Fondo de Investigación Sanitaria (FIS), Ministry of Science and Innovation, Spain, and the Ajuts per donar Suport als Grups de Recerca de Catalunya sponsored by the Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR), Generalitat de Catalunya, Spain.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. Disclosure
  9. References
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