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. 2015 Aug 31;182(2):162–172. doi: 10.1111/cei.12689

Effect of interferon-β1b on CXCR4-dependent chemotaxis in T cells from multiple sclerosis patients

T Wostradowski *,†, V Gudi *, R Pul *, S Gingele *, J A Lindquist ‡,§, M Stangel *,†,1,✉, S Lindquist *,¶,1
PMCID: PMC4608506  PMID: 26212126

Abstract

Multiple sclerosis (MS) is an inflammatory, demyelinating and neurodegenerative disease triggered by infiltration of activated T cells into the central nervous system. Interferon (IFN)-β is an established, safe and effective treatment for patients with relapsing–remitting MS (RRMS). The cytokine can inhibit leucocyte infiltration into the central nervous system; however, little is known about the precise molecular mechanisms. Previously, in vitro application of IFN-β1b was shown to reduce CXCL12/CXCR4-mediated monocyte migration. Here, we analysed the effects of IFN-β1b on CXCR4-dependent T cell function. In vitro exposure to IFN-β1b (1000 U/ml) for 20 h reduced CXCR4-dependent chemotaxis of primary human T cells from healthy individuals and patients with RRMS. Investigating the IFN-β1b/CXCR4 signalling pathways, we found no difference in phosphorylation of ZAP70, ERK1/2 and AKT despite an early induction of the negative regulator of G-protein signalling, RGS1 by IFN-β1b. However, CXCR4 surface expression was reduced. Quantitative real time-PCR revealed a similar reduction in CXCR4-mRNA, and the requirement of several hours' exposure to IFN-β1b supports a transcriptional regulation. Interestingly, T cells from MS patients showed a lower CXCR4 expression than T cells from healthy controls, which was not reduced further in patients under IFN-β1b therapy. Furthermore, we observed no change in CXCL12-dependent chemotaxis in RRMS patients. Our results demonstrate clearly that IFN-β1b can impair the functional response to CXCR4 by down-regulating its expression, but also points to the complex in vivo effects of IFN-β1b therapy.

Keywords: CXCL12, CXCR4 expression, interferon-β1b, migration, primary human T cells

Introduction

Infiltration of activated autoreactive T cells into the central nervous system (CNS) is a key event in the pathogenesis of multiple sclerosis (MS) 1,2. To prevent a rapid accumulation of new inflammatory MS lesions, interferon (IFN)-β has been used successfully in the treatment of relapsing–remitting MS (RRMS) for more than two decades 3,4. However, the cellular and molecular mechanisms that convey the immunomodulatory effect of IFN-β are still not completely understood 5–7. Several modes of action have been proposed, including suppression of T cell activation and proliferation 8, reduction of leucocyte migration across the blood–brain barrier (BBB) by the inhibition of matrix metalloproteinases, shifting of the cytokine profile and BBB modifications 9–12. For the transmigration of human leucocytes into the CNS, the chemokine CXCL12, also known as stromal cell-derived factor 1 alpha (SDF1α) has been identified as a key molecule 13,14. CXCL12 binds to CXCR4 and CXCR7, both G-protein-coupled receptors. While CXCR7 is a non-signalling receptor, modulating the localization of CXCL12 by internalization and translocation 15,16, CXCR4 signalling is crucial to mediate CXCL12 function in immune cells and neurons 16. CXCR4 is broadly expressed and is involved in the development and function of multiple tissues, including the immune and the nervous system. In the peripheral immune system, CXCL12 is a strong chemoattractant for T cells, B cells and monocytes, which express CXCR4 17. Further functions of CXCL12/CXCR4 signalling include supporting the survival of mature lymphocytes and the generation of memory T cells 18. In different inflammatory or autoimmune conditions, CXCL12/CXCR4 signalling was shown to enhance the inflammatory infiltration of lymphocytes. In rheumatoid arthritis, for example, CXCL12/CXCR4 signalling was shown to modulate T cell accumulation in synovial tissue 19. In the CNS, constitutive CXCL12 expression was demonstrated on blood vessel walls in brain parenchyma. In both active and chronic inactive multiple sclerosis lesions, CXCL12 was elevated on blood vessels and induced in astrocytes 20. In RRMS patients CXCL12 was found to be increased in the cerebrospinal fluid (CSF) in the absence of BBB disruption, while patients with other inflammatory neurological diseases showed high CXCL12 concentrations in the CSF, but also had severe BBB disruption. Interestingly, patients with progressive multiple sclerosis, where CNS inflammation is believed to be driven by a local immune response rather than infiltrating lymphocytes 20, showed no significant increase in CXCL12 levels in the CSF.

Binding of CXCL12 to CXCR4 initiates several signal transduction pathways, most importantly the activation of the phosphoinositide phospholipase C/inositol trisphosphate (PLCγ-IP3) pathway, the activation of several mitogen-activated protein kinases (MAPK), including p44/42 MAPK ERK1/2 21, phosphoinositide 3-kinase/protein kinase B (PI3K-AKT) 21,22 and the Janus kinase/signal transducer and activator of transcription (JAK/STAT) pathway 23. Signalling cascades triggered by the type I interferon (IFN) receptor could interfere with CXCR4 signalling via shared mediators, e.g. ZAP70, PI3K-AKT or ERK1/2 24,25. We therefore hypothesized that IFN-β1b could modulate CXCR4-mediated effects; namely, the attraction of lymphocytes by CXCL12 to sites of neuroinflammation. Indeed, a reduced in vitro migration of human monocytes stimulated with CXCL12 after IFN-β1b pretreatment has been demonstrated previously 26. Given the importance of T cells for lesion development in MS, we investigate here whether IFN-β can intercept the CXCR4 signalling cascade and alter functional CXCR4-dependent responses in T cells.

Materials and methods

Patient recruitment

MS patients were diagnosed with RRMS or clinically probable MS according to the revised McDonald criteria 27. Patients were recruited only if they were free of exacerbations for at least 1 month before blood collection. The patient cohort had not received immunosuppressants for at least 3 months and corticosteroids treatment for 1 month prior to and during the study. MS patients not currently receiving any immunomodulatory treatment were compared to those treated with IFN-β1b for at least 6 months (Betaferon®; Bayer Healthcare, Leverkusen, Germany) and to healthy individuals (Table1). The blood samples were collected from MS patients who had been treated with IFN-β1b within the last 16–18 h after the IFN-β injection. The study was reviewed and approved by the Ethics Committee of the Hannover Medical School (ethics committee vote no. 1322-2012). All patients gave written informed consent.

Table 1.

Biometric data of all volunteers included in this study.

Total number of relapse Duration of IFN-β1b treatment in years
n m/f Age in years Duration of MS in years Before treatment Under treatment
Healthy controls 20 3/17 34·1 ± 11·3
MS patients without DMT 18 6/12 35·2 ± 11·3 6·5 ± 9·4 2·2 ± 2·3
IFN-β1b-treated MS patients 10 3/7 41·4 ± 9·5 8·9 ± 8·4 3·1 ± 3·0 1·5 ± 1·4 4·5 ± 5·2
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m = male; f = female. Mean values ± SD are shown.

Preparation of T cells and cell culture

Peripheral blood mononuclear cells (PBMCs) were isolated from donors using a Biocoll separating solution (Biochrom AG, Berlin, Germany) by density gradient centrifugation (14°C, 474 g, 25 min, without brake). After centrifugation, PBMCs were isolated from the interphase of the resulting gradient. Cells were washed three times in phosphate-buffered saline (PBS) (Life Technologies, Darmstadt, Germany) and counted. T cells were purified from the interface of PBMCs by negative selection using the autoMACS magnetic separation system, according to the manufacturer's instructions (Miltenyi Biotec, Bergisch Gladbach, Germany). Purity of the separated T cells (> 92%) was analysed by flow cytometry using FITC-conjugated anti-CD3ε (UCHT1), PE-conjugated anti-CD14 (HCD14) and APC-conjugated anti-CD19 (HIB19) (all from BioLegend, San Diego, CA, USA).

T cells were resuspended in RPMI-1640 medium containing 0·3 g/l L-glutamine (Life Technologies, Darmstadt, Germany), 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin solution (all Biochrom AG, Berlin, Germany).

Stimulation of primary human T cells in vitro

For IFN-β1b pretreatment, T cells were incubated with or without IFN-β1b (Bayer Healthcare, Leverkusen, Germany) at a final concentration of 1000 U/ml for 1 h or 20–24 h at 37°C, 5% CO2 in a humidified atmosphere.

For Western blot analysis of CXCR4-triggered signalling events, untreated and IFN-β1b pretreated cells (2 × 106 cells/sample) were harvested and stimulated for 0 (control), 1, 3 and 10 min with a 10 nM final concentration of CXCL12 (PeproTech, Hamburg, Germany) in serum-free RPMI-1640 medium (Life Technologies, Darmstadt, Germany) with or without IFN-β1b at 37°C. Reactions were stopped by addition of ice-cold PBS. After stimulation, the cells were pelleted for 2 min at 2655 g and lysed in an equal volume (40 µl) of lysis buffer (50 mM HEPES pH 7·4; 100 mM NaCl; 1% Nonidet P-40; 5 mM EDTA; 1% n-dodecyl-β-D-maltoside; 1 mM sodium orthovanadate; 50 mM NaF and 10 mM sodium pyro-phosphate) supplemented with 1% protease inhibitor cocktail (Roche, Applied Science, Mannheim, Germany) for 30 min on ice. Samples were centrifuged at 10 000 g for 15 min at 4°C. The supernatant was then heated for 5 min at 95°C.

Gel electrophoresis and Western blotting

Equal amounts of cell lysate were separated by SDS-PAGE using 12% polyacrylamide gels. After electrophoresis, total cell proteins were transferred onto polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA) and incubated for 1 h at room temperature in blocking solution 5% non-fat milk made in Tris-buffered saline plus Tween 20, (TBST), washed in TBST and incubated with primary antibodies overnight at 4°C. The following primary antibodies were used: anti-phospho(p)-T202/Y204 ERK1/2 (#9101), anti-total ERK1/2 (#9107), anti-phospho-S473 AKT (#9271), anti-AKT (9272), anti-phospho-Y701 STAT-1 (#9171), anti-phospho-Y319 ZAP-70/Y352 SYK (#2701) (all Cell Signalling Technology, Danvers, MA, USA), and anti-C-23 GRB2 (sc-255) (Santa Cruz Biotechnology, Dallas, TX, USA) and anti-human RGS1 antibody (#PA5-29579) (Pierce Biotechnology, Rockford, IL, USA). After washing followed incubation with the appropriate horseradish peroxidase (HRP)-coupled goat anti-rabbit or goat anti-mouse secondary antibodies (all R&D Systems, Wiesbaden-Nordenstadt, Germany). Proteins were visualized by enhanced chemiluminescence (ECL; Pierce Biotechnology, Illinois, USA; Millipore, Massachusetts, USA) after treatment with secondary antibody using the ChemoCam system (intas; Science Imaging Instruments GmbH, Göttingen, Germany), according to the manufacturer's instructions. Quantification of protein levels by densitometry was conducted on acquired images using LabImage 1D software (Kapelan Bio-Imaging Solutions, Leipzig, Germany).

Transwell migration

CXCL12-dependent cell migration was assessed using 5-µm-pore Transwell filter membranes (Corning Inc., Corning, NJ, USA). The untreated or 20 h IFN-β1b (1000 U/ml) pretreated cells were washed and resuspended at 5 × 106 cells/ml in migration medium (RPMI-1640; 1% BSA; 20 mM HEPES, pH 7·4). Migration medium with and without IFN-β1b was loaded into the lower wells of a 24-well plate. The chemokine CXCL12 was then added to a 10 nM final concentration. Transwell inserts contained 50 µl of cell suspension (2·5 × 105; untreated or IFN-β1b pretreated) and 50 µl of migration medium. The plate was incubated at 37°C for 1 h; the inserts were then removed carefully and the migrated cells were collected from the lower wells and counted by flow cytometry 28. All migration experiments were carried out in duplicate and repeated independently several times, as indicated. Results are shown either as percentage of migration in relation to the initial numbers of cells applied or as migration of cells relative to the stimulated and untreated control (+CXCL12, without IFN-β1b).

Flow cytometry analysis

The fluorescent staining was performed on single-cell suspensions of non-permeabilized cells in PBS. Total T cells (5 × 105 cells/sample) were resuspended in 100 µl of PBS after centrifugation (352 g, 10 min). The suspensions were incubated for 30 min on ice with the fluorescent-labelled antibodies, FITC-conjugated anti-CD3ε and APC-conjugated anti-CD184 (CXCR4; 12G5) or APC-conjugated IgG2a, κ as isotype control for CXCR4 (MOPC-173) (all from BioLegend). After incubation cells were washed, pelleted by centrifugation (352 g, 10 min) and resuspended in 250 µl of PBS. Cells were analysed immediately with FACS Calibur and CellQuest software (BD Biosciences). Dead cells were excluded by propidium iodide staining.

Quantitative real-time polymerase chain reaction (RT-PCR)

The change in CXCR4-mRNA expression was analysed after 6 and 24 h of activation of primary human T cells (total amount: 3·7 × 106−23·5 × 106) from healthy donors with IFN-β1b (100, 250, 500 or 1000 U/ml).

Total RNA was isolated from primary human T cells using the RNeasy® Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's instructions. The RNA concentration was measured using a NanoDrop 1000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA). For the synthesis of cDNA we used the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). Real-time quantitative PCR analysis was performed using the StepOne™ real-time PCR system and appropriate SensiFAST™ Probe Hi-ROX Kit (BIO-82005; Applied Biosystems, Foster City, USA). The experiments were performed with cDNA from 250 ng total RNA and all primers were exon-spanning (TaqMan® Gene Expression Assay, inventoried, CXCR4 Hs00237052_m1). The 2−ΔΔCT method was used to determine differences in expression of the CXCR4 gene between untreated and IFN-β1b pretreated T cells 29. Changes in the mRNA expression levels were calculated after normalization to the housekeeping gene hypoxanthine-guanine phosphoribosyl-transferase in comparison to untreated control.

Statistical analysis

All analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA, USA). Comparison of two samples was performed using Student's t-test. Multiple samples were evaluated using repeated measures ANOVA combined with Bonferroni's multiple comparison or Newman–Keuls post-hoc test, as indicated. All data were calculated as arithmetic means ± standard deviation (SD) and the significance level was set at P < 0·05.

Results

IFN-β1b reduces CXCL12-dependent migration of primary human T cells

The migration of activated T cells into the CNS is critical for initiating and sustaining the pathology of MS, but little is known about the role of IFN-β in the regulation of immune cell migration 5. To address this point, we studied the effect of recombinant IFN-β1b on chemokine-dependent T cell migration in vitro using a Transwell migration assay. We pretreated freshly isolated human T cells with IFN-β (1000 U/ml) for a time-period of 1 or 20 h and analysed CXCL12-induced migration (Fig. 1). The migration of cells relative to the stimulated and untreated control (+CXCL12, without IFN-β1b) was reduced by 42·0 ± 17·6% (mean ± SD) after IFN-β1b pretreatment for 20 h (Fig. 1b). Pretreatment with IFN-β (1000 U/ml) for 1 h (Fig. 1a) or exposure to lower doses of IFN-β1b (100 and 250 U/ml) for 20 h were not sufficient to inhibit T cell migration (data not shown).

Figure 1.

Figure 1

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Interferon (IFN)-β1b reduces CXCL12-dependent migration of purified T cells in vitro. T cells were isolated from healthy individuals and pretreated with IFN-β1b (1000 U/ml) for 1 h (a, n = 6) or 20 h (b, n = 18). T cell transmigration across an artificial barrier in response to CXCL12 (10 nM)-stimulation was determined as described previously 28. Data are presented as migration of cells relative to the stimulated and untreated control (+CXCL12, without IFN-β1b) expressed as the mean ± standard deviation (SD) and were analysed by repeated measures ANOVA and Bonferroni's multiple comparison post-hoc test. Significant effects are indicated by asterisk (***P < 0·001).

IFN-β1b does not affect CXCR4-dependent signalling molecules

Binding of the chemokine CXCL12 to the CXCR4 receptor induces a number of G protein-mediated signalling events in T cells. RGS family proteins control G-protein signalling by shortening the half-life of the active GTP-bound alpha subunits. Induction of one member of this family, RGS1, via IFN-β was found responsible for a reduced CXCL12-dependent migration of primary human monocytes 26. Interestingly, IFN-β1b was shown to induce RGS1 gene expression in PBMCs as well as in monocytes pretreated 4 or 18 h 26. To explore whether the observed reduction in CXCL12-mediated T cell migration by in vitro exposure to IFN-β could be explained by induction of RGS1 protein expression in primary human T cells, we stimulated T cells from healthy individuals with IFN-β1b (1000 U/ml) for either 1 or 24 h. PBMCs were stimulated for 24 h as positive control. Western blot analysis shows a strong induction of RGS1 by IFN-β, similar to the results of Tran and colleagues 26. However, contrary to the situation reported in PBMCs 26, RGS1 protein was already induced after 1 h pretreatment with IFN-β1b in primary human T cells (Fig. 2).

Figure 2.

Figure 2

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Interferon-β1b pretreatment induces RGS1 protein expression. Peripheral blood mononuclear cells (PBMCs) (n = 3) or highly enriched T cells (n = 5) were obtained from healthy individuals and were pretreated with IFN-β1b (1000 U/ml) for either 1 h or 24 h, as indicated. (a) RGS1 expression was determined via Western blot analysis. (b) All densitometric analysed data were normalized to β-actin and expressed as the mean ± standard deviation (SD). Data were analysed by repeated measures ANOVA and Newman–Keuls post-hoc test. Significant effects are indicated by asterisk (**P < 0·01; *P < 0·05).

Quantitative analysis of five independent samples revealed that induction was less strong after 24 h pretreatment (Fig. 2b). Given the early induction of RGS1, it is unlikely that direct negative regulatory effects of RGS1 explain the reduction of CXCL12/CXCR4-dependent migration in primary human T cells by IFN-β1b after only 20 h pretreatment.

To understand more clearly the observed IFN-β1b effect, we examined CXCR4 signalling more closely. The CXCL12/CXCR4 chemokine–chemokine receptor pair induces a number of signalling pathways, in particular a strong activation of MAPK p44/42 ERK1/2, which is important for the induction of gene expression. ERK1/2 activation by CXCL12 requires the presence of the Syk family kinase ZAP70. The phosphorylation of the serine/threonine kinase AKT is also triggered after activation of the CXCR4 receptor. In addition to the JAK/STAT pathway, IFN-β signalling also utilizes ZAP70, ERK1/2 and AKT. Therefore, we investigated potential signalling cross-talk of IFN-β with CXCR4 in T cells from healthy controls after 24 h of IFN-β1b pretreatment. Stimulation with CXCL12 led to a robust phosphorylation of AKT and ERK, but activation of both key signalling mediators was not affected by IFN-β pretreatment (Fig. 3). Interestingly, we did not observe a reduction of ERK1/2 phosphorylation after 24 h IFN-β1b pretreatment. As a control for the activity of IFN-β in vitro, a strong induction of STAT-1-phosphorylation was found at all time-points (Fig. 3a).

Figure 3.

Figure 3

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Interferon (IFN)-β1b pretreatment for 24 h does not modify CXCL12/CXCR4-dependent signalling. IFN-β1b did not enhance CXCL12/CXCR4-dependent phosphorylation of ERK and AKT or ZAP70. Cells were incubated with or without 1000 U/ml IFN-β1b for 24 h and were stimulated with CXCL12 for the time indicated. (a) Cell lysates were blotted onto membranes and probed with the corresponding antibodies, then analysed by using Western blot. Phospho-antibodies against AKT and ERK1/2 were used to quantify changes in phosphorylation levels compared to non-phosphorylated AKT, ERK. GRB2 was used as a loading control. (b) Summary of three independent experiments as in (a) expressed as the mean ± standard deviation (SD). (c) Summary of nine independent experiments as in (a) expressed as the mean ± SD. Repeated measures ANOVA showed no statistically significant differences between untreated or IFN-β1b pretreated CXCL12-stimulated samples.

IFN-β1b pretreatment decreases CXCR4 expression in vitro

As IFN-β1b pretreatment for 24 h did not affect CXCR4-dependent signalling, we next analysed whether CXCR4 receptor expression was altered. CXCR4 expression was tested by flow cytometry using non-permeabilized human T cells isolated from healthy individuals with or without in vitro IFN-β1b pretreatment for 24 h (Fig. 5b). IFN-β1b pretreatment reduced the mean surface expression of CXCR4 on T cells from healthy individuals (by 41·8 ± 10·8%). We could not detect a reduction in CXCR4 surface expression after 1 h incubation with IFN-β1b (data not shown). Next, we analysed whether a regulation occurs at the level of gene expression. The CXCR4-mRNA was analysed by quantitative RT-PCR (Fig. 4a,b). CXCR4-mRNA was reduced to approximately 50% after IFN-β1b pretreatment (1000 U/ml) for 6 h (P < 0·05). After pretreatment with lower doses (100–500 U/ml) for 6 h a trend to reduced CXCR4-mRNA was seen, but did not reach significance (Fig. 4b).

Figure 5.

Figure 5

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CXCR4 surface expression of T cells from healthy controls (HC) and MS patients after IFN-β1b treatment in vivo or in vitro. Peripheral blood T cell preparations obtained from healthy individuals (n = 16) or from MS patients without disease-modifying treatment (DMT) (n = 15) or IFN-β1b-treated MS patients (n = 9), followed by a minimum of 20 h incubation with or without IFN-β1b (1000 U/ml). Non-permeabilized cells were stained with CD3/CXCR4 antibodies and the mean fluorescence intensity (MFI) was determined by flow cytometry. The values were expressed as the mean ± SD and analysed by one-way ANOVA with Bonferroni's multiple comparison test (a) or paired Student's t-test (b–d). Significant effects are indicated by asterisk (***P < 0·001; *P < 0·05). (a) Mean fluorescence intensity of CXCR4 from patients without DMT and IFN-β1b-treated patients is depicted with data from healthy controls as shown in (b). MS patients without DMT show a lower CXCR4 expression than healthy controls. There is no significant difference in IFN-β-treated patients. (b) IFN-β1b reduces expression of CXCR4 on primary human T cells from healthy individuals, as shown in a reduced mean fluorescence intensity (MFI) of CXCR4. (c) CXCR4-MFI of T cells from MS patients without DMT for 20–24 h with or without IFN-β1b. The data in the left column (0 h IFN-β1b pretreatment) are also depicted in (a), middle column. (d) CXCR4-MFI of T cells from MS patients under IFN-β1b therapy for 20–24 h with or without IFN-β1b. The data in the left column (0 h IFN-β1b pretreatment) are also depicted in (a), right column.

Figure 4.

Figure 4

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Interferon (IFN)-β1b reduces gene expression of CXCR4 on primary human T cells from healthy individuals. (a,b) The change of mRNA expression of chemokine receptor CXCR4 from healthy controls (n = 4) were analysed after time- and dose-dependent activation of either 6 or 24 h and either 1000 U/ml or 100, 250 or 500 U/ml IFN-β1b pretreatment. Significant differences between untreated or IFN-β1b pretreated samples for the duration of pretreatment was calculated by non-parametric Mann–Whitney test. Significant effects are indicated by asterisk (*P < 0·05).

In contrast to the results after 6 h IFN-β pretreatment, the reduction of the CXCR4 transcripts after 24 h IFN-β pretreatment compared to 0 h was not significant (Fig. 4a,b).

IFN-β1b pretreatment affects cell migration and CXCR4 expression in MS patients

So far, we have demonstrated a role of IFN-β1b in functional CXCL12-dependent responses in T cells isolated from healthy individuals. Next, we analysed the effects of IFN-β on cell migration and CXCR4 expression on T cells from RRMS patients.

Flow cytometric analysis revealed that the mean surface CXCR4 expression is reduced unexpectedly on T cells of RRMS patients without immunomodulatory treatment in vivo. In contrast to in vitro exposure to IFN-β1b, a trend towards increased CXCR4 expression could be observed on T cells from IFN-β-treated MS patients in vivo, but this did not reach significance (Fig. 5a). When comparing CXCL12-stimulated migration of T cells isolated ex vivo from patients either under IFN-β1b therapy or without disease-modifying treatment (DMT), no difference in the number of migrating cells could be observed (Fig. 6a).

Figure 6.

Figure 6

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CXCL12/CXCR4-dependent migration of T cells from multiple sclerosis (MS) patients after IFN-β1b treatment in vivo and in vitro. Peripheral blood T cell preparations obtained from healthy individuals (n = 16), from MS patients without DMT (n = 15), or IFN-β1b-treated MS patients (n = 9), followed by 20–24 h incubation with or without IFN-β1b (1000 U/ml). CXCL12-dependent migration was tested as described previously. (a) T cells from patients without DMT or IFN-β1b-treated patients were cultured for 20–24 h without IFN-β1b. Data are presented as percentage of migration to the initial numbers of cells and were analysed by one-way repeated measures ANOVA. (b) T cells from relapsing–remitting MS (RRMS) patients without DMT were cultured for a minimum 20 h with or without IFN-β1b. Data are shown as migration of cells relative to the stimulated control (+CXCL12, without IFN-β1b) and were analysed by repeated measures ANOVA and Bonferroni's multiple comparison test. (c) T cells from RRMS patients under IFN-β1b therapy were cultured for a minimum 20 h with or without IFN-β1b. Data are as migration of cells relative to the stimulated control (+CXCL12, without IFN-β1b) and were analysed by repeated measures ANOVA and Bonferroni's multiple comparison test. Significant effects are indicated by asterisk (***P < 0·001; **P < 0·01).

When T cells from MS patients were subjected to IFN-β1b pretreatment in vitro for 24 h, a reduction in the mean CXCR4 surface expression of 33·5 ± 13·7% (mean ± SD) was observed for MS patients without DMT (Fig. 5c) and of 37·0 ± 12·1% (mean ± SD) in T cells from IFN-β1b treated patients (Fig. 5d), thus confirming our initial finding that IFN-β1b reduces CXCR4 expression on T cells. Similarly, T cell migration was also reduced after IFN-β1b pretreatment for 20–24 h for MS patients without DMT (by 30·4 ± 17·7%) and for IFN-β1b-treated MS patients (by 29·1 ± 28·5%) (Fig. 6c,d).

Discussion

This study demonstrates that recombinant IFN-β1b treatment reduces CXCR4 expression and CXCR4-dependent migration of primary human T cells in vitro. CXCR4 expression on T cells was found reduced in MS patients compared to healthy controls, while IFN-β1b therapy improved CXCR4 expression contrasting with the effect of IFN-β1b treatment in vitro.

High-dose IFN-β has been used safely and successfully for the past 20 years to reduce the number of relapses and brain lesions and to slow disability progression in RRMS patients 30. In particular, the number of Gadolinium-enhancing lesions, the radiological sign for BBB disruption, is reduced by 85–90% within a month of starting IFN-β therapy 31, indicating a robust effect on leucocyte transmigration into the CNS.

Several mechanisms have been suggested to mediate this effect of IFN-β, e.g. reduction in the expression of metalloproteinases and the increase of soluble VCAM1 in serum, which is believed to inhibit VLA-4-mediated leucocyte adhesion by competitive binding 12. Together with adhesion molecules, chemokines govern leucocyte transmigration into the CNS. Among the C-X-C-chemokines, CXCL12 and its receptor CXCR4 appear to play distinct roles in the development of active lesions 20,32, supporting the entry of activated lymphocytes into the CNS, but also retaining them in the perivascular cuff 33.

A link between IFN-β and CXCR4 signalling was first provided by a study on primary human mononuclear cells 26. Incubation with IFN-β (1000 U/ml) for 20 h was shown to inhibit CXCL12- and MCP1-driven migration of primary human monocytes and the human monocytic cell line THP1 in vitro. CXCR4 surface expression was reported unaltered, and up-regulation of a negative regulator of G-protein signalling, RGS1, was suggested to mediate this effect of IFN-β. Indeed, RGS1-mRNA was shown to be induced in PBMC by exposure to IFN-β (1000 U/ml) for 4 h in culture. This RGS1-mRNA induction was highest in primary human monocytes and lower in T cells and B cells 26. The study further demonstrated that RGS1-protein was induced in human PBMC by exposure to IFN-β for 20 h in vitro. MS patients under established IFN-β1b therapy were subjected to a washout period of 64 h and PBMC were analysed prior to administration of a therapeutic IFN-β1b dose (8 M IU, 250 µg/ml) 4, 18 and 42 h thereafter. A rise in RGS1-mRNA was shown consistently, albeit with high interindividual variability 26.

We asked whether IFN-β could specifically inhibit CXCR4-dependent migration of T cells. As very little IFN-β crosses the BBB, most direct effects will be caused via circulating immune cells 34. Among these, T cells are the most important in triggering the development of new lesions, while monocyte infiltration and activation are thought to be secondary.

Studying highly purified primary human T cells, we could extend the findings of Tran et al., showing a robust inhibition of CXCL12-driven transmigration by exposure to IFN-β1b for 20 h. We then investigated whether a direct signalling cross-talk between the type I IFN receptor and CXCR4 could account for this effect. However, a short exposure to IFN-β1b for 1 h in vitro was not sufficient to affect CXCL12-driven transmigration, arguing for the need of a transcriptional event. We confirmed, in purified T cells, that RGS1-protein levels are increased; however, RGS1 was already increased strongly after 1 h of IFN-β1b exposure. This might be due in part to mRNA-independent effects on protein stability, but makes it less likely that RGS1 is responsible for the effect on CXCR4-dependent migration, which was seen only after > 20 h of IFN-β1b exposure. We therefore studied whether major nodes in the CXCR4 signalling cascade were affected by 24 h exposure to IFN-β1b in vitro, but we found no effect on the phosphorylation of AKT and p44/42 MAPK or ZAP70. We therefore conclude that the effect of IFN-β1b on CXCR4-dependent migration is not due to interference with downstream chemokine receptor signalling and cannot be explained by the induction of RGS1 in primary human T cells. IFN-β as a member of the type I IFN family of cytokines is physiologically important for the innate and acquired immunity against infectious pathogens, including viral proteins and bacterial components. Apart from modulating the trafficking of immune cells, CXCR4 acts as a co-receptor for entry of HIV strains. Regulation of CXCR4 surface expression may be part of the anti-viral activity of type I IFNs. Indeed, a downregulation of CXCR4-mRNA in human PBMC by in vitro exposure to IFN-α and IFN-γ has been demonstrated 35, and mice deficient for IFN-β show increased CXCR4 expression on CD4+ T cells 36. Therefore, we asked whether altered CXCR4 surface expression could explain the inhibition of CXCL12-mediated T cell migration. We found that exposure to IFN-β1b in vitro for 20–24 h robustly reduced CXCR4 surface expression. The mechanism of this IFN-β effect is not clear; however, we could show a downregulation of CXCR4-mRNA, correlating with the reduction in surface protein and confirming a cDNA microarray of PBMC treated ex vivo with IFN-β 37. Interestingly, this study shows dynamic expression of IFN-induced genes within the first months of IFN-β therapy; in particular, a peak in MxA (myxovirus resistance protein A) expression after 2 months and a decline thereafter. Adaptive changes may explain that in contrast to the in vitro data, intraindividual comparison of gene expression in PBMC from RRMS patients before and after 2 years of IFN-β therapy (IFN-β1a) found increased CXCR4-mRNA in IFN-β-treated patients 38. Similarly, we found a trend to increased CXCR4-protein surface expression in RRMS patients under established IFN-β1b therapy. Surprisingly, we found that CXCR4 expression was reduced in MS patients without DMT compared to healthy controls and IFN-β1b therapy improved CXCR4 surface protein. The finding of reduced CXCR4 surface expression in RRMS patients conflicts with the findings in MS lesions, where an increase in CXCR4 is found particularly at the lesion edge 39. Two hypotheses could explain a reduced CXCR4 surface expression in peripheral T cells of RRMS patients. First, the peripheral population analysed might be different from the population infiltrating into the CNS. Secondly, the reduced CXCR4 expression could be secondary to activation after ligand binding and subsequent downregulation of the surface receptor. It is intriguing to speculate that the increased surface expression found in patients under established IFN-β therapy might be an indirect effect, due perhaps to decreased adhesion to the BBB (via effects on adhesion molecules, such as VLA-4), where most CXCL12 ligand is presented, and lack of receptor internalization. Naturally, the difference between the in vitro and in vivo effects of recombinant IFN-β1b might also be due simply to dose-dependent differential effects on CXCR4. Data on in vivo concentrations of IFN-β1b vary, but levels appear to not exceed 250 U/ml 40, while 1000 U/ml were used in most in vitro studies. While, using doses of 100 and 250 U/ml of IFN-β1b, we failed to observe an effect on CXCR4-dependent migration in vitro, our dose–response curve suggested that doses between 100 and 500 U/ml IFN-β1b are sufficient to downregulate CXCR4-mRNA in vitro. We hypothesize that in vitro lower doses of IFN-β might trigger some, but not all, of the relevant signalling events to inhibit CXCL12-dependent chemotaxis. The threshold for CXCR4 signalling is adjusted by signalling cross-talk with other receptors, e.g. the T cell receptor (TCR) 41 or, inferring from data in neuronal stem cells 42, by BDNF. In vivo, additional factors triggered by IFN-β, such as enhanced BDNF expression 43, together with its effect on CXCR4-mRNA expression, could lead to a functionally relevant effect in RRMS patients.

Limitations of our study include the small sample size and the purely cross-sectional design. Longitudinal analysis before and after establishing IFN-β therapy as well as a defined time–course after individual injections, and comparison of CXCR4-mRNA with receptor surface expression in IFN-β-treated patients and comparative analysis of cells in serum and CSF, would help to improve our understanding of the in vivo effect of IFN-β on CXCR4.

To summarize, we observed a consistent direct effect of IFN-β in vitro downregulating CXCR4-mRNA and surface protein in primary human T cells in healthy controls and RRMS patients alike. The complex and indirect in vivo effects of IFN-β therapy in RRMS patients require further studies.

Acknowledgments

This study was funded partly by Bayer HealthCare Pharmaceuticals, and the authors thank Dr Glaser (Bayer HealthCare, Leverkusen) for support. We thank Dr Kloth (Department of Pharmacology, Hannover Medical School) and Prof. Dr Claus (Department of Neuroanatomy, Hannover Medical School) for technical advice. We thank Dr med. Skripuletz, Dr med. Alvermann, Dr med. Sühs and Mrs Hache-Janning for the assistance in recruiting patients. The authors sincerely thank the individuals who volunteered to be subjects in this study. J. A. L. was a member of the Magdeburg Centre for Systems Biology (MACS) and the SYBILLA consortium (European Union 7th Frame Program).

Author contributions

M. S. and S. L. designed the project, supervised and co-ordinated the work, analysed and interpreted data as well as participated in writing the manuscript. T. W. co-ordinated and performed experiments, collected and analysed data and wrote the manuscript. J. A. L. helped to design the experiments and to interpret data. V. G. performed real-time PCR. R. P. and S. G. were involved in patient recruitment and collection of the patients' data. All authors have read, commented on and approved the final manuscript.

Disclosure

This research work was supported partly by Bayer HealthCare. The sponsor was not involved in data collection or analysis, drafting the manuscript or the decision to publish. This work is part of the doctoral thesis of T. W. at the University of Veterinary Medicine Hannover. T. W. has received a travel stipend for attending an immunology congress. V. G. has no conflicts of interest. R. P. has received research funds from TEVA Pharmaceutical Industries and travel grants from Baxter, Bayer HealthCare, Biogen, Novartis, Sanofi-Aventis and Merck Serono. S. G. and J. A. L. have no conflicts of interest. M. S. has received honoraria for scientific lectures or consultancy from Bayer Healthcare, Biogen Idec, Baxter, CSL Behring, Grifols, Merck-Serono, Novartis, Sanofi-Aventis and Teva. His institution received research support from Bayer Healthcare, Biogen Idec, Merck-Serono, Novartis and Teva. S. L. has received travel grants from Bayer Healthcare, Novartis and Genzyme and honoraria for scientific lectures and consulting from Bayer Healthcare, Genzyme and Novartis.

Supporting Information

Additional Supporting information may be found in the online version of this article at the publisher's web-site:

Fig. S1. CXCR4 surface expression of CD4+ and CD8+ T cell subsets from healthy individuals after interferon (IFN)-β1b treatment in vitro. Peripheral blood T cell preparations obtained from healthy individuals (n = 4) followed by 24 h incubation with or without IFN-β1b (1000 U/ml). Non-permeabilized cells were stained with CD3/CXCR4 antibodies and with antibodies to determine CD4 or CD8 T cell subset. The mean fluorescence intensity (MFI) was obtained by flow cytometry. The values were expressed as the mean ± standard deviation (SD) and analysed by paired Student's t-test. Significant effects are indicated.

cei0182-0162-sd1.tif (371.6KB, tif)

Fig. S2. Interferon (IFN)-β1b pretreatment induces RGS1 protein expression in highly enriched T cells. Peripheral blood mononuclear cells (PBMC) were obtained from healthy individuals (n = 4). After MACS cell separation as described in the Methods section and cell sorting (MoFlo XDP Cell Sorter; Beckman Coulter) we obtained highly purified T cells (purity > 99.9%). These T cells were pretreated with IFN-β1b (1000 U/ml) for either 1 or 24 h, as indicated. (a) RGS1 expression was determined via Western blot analysis. For Western blot analysis, equal amounts of protein were subjected to electrophoresis in 12% SDS-PAGE gels, transferred to a PVDF membrane and blocked with TBS 1× supplemented with 0·1% Tween 20/5% non-fat dry milk. The PVDF membrane was incubated overnight at 4°C with a rabbit RGS1 antibody (rabbit, polyclonal; Pierce) at a 1:1000 dilution, washed and incubated with a secondary HRP-conjugated anti-rabbit antibody (goat; R&D Systems) at a 1:3000 dilution. After enhanced chemoluminescence reaction (SuperSignal West Femto system; Pierce), the bands were visualized. (b) All densitometric analysed data were normalized to α-tubulin and expressed as the mean ± standard deviation (SD). Comparison of each sample was performed using paired Student's t-test. Significant effects are indicated by asterisk (**P < 0·01).

cei0182-0162-sd2.tif (271.1KB, tif)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Fig. S1. CXCR4 surface expression of CD4+ and CD8+ T cell subsets from healthy individuals after interferon (IFN)-β1b treatment in vitro. Peripheral blood T cell preparations obtained from healthy individuals (n = 4) followed by 24 h incubation with or without IFN-β1b (1000 U/ml). Non-permeabilized cells were stained with CD3/CXCR4 antibodies and with antibodies to determine CD4 or CD8 T cell subset. The mean fluorescence intensity (MFI) was obtained by flow cytometry. The values were expressed as the mean ± standard deviation (SD) and analysed by paired Student's t-test. Significant effects are indicated.

cei0182-0162-sd1.tif (371.6KB, tif)

Fig. S2. Interferon (IFN)-β1b pretreatment induces RGS1 protein expression in highly enriched T cells. Peripheral blood mononuclear cells (PBMC) were obtained from healthy individuals (n = 4). After MACS cell separation as described in the Methods section and cell sorting (MoFlo XDP Cell Sorter; Beckman Coulter) we obtained highly purified T cells (purity > 99.9%). These T cells were pretreated with IFN-β1b (1000 U/ml) for either 1 or 24 h, as indicated. (a) RGS1 expression was determined via Western blot analysis. For Western blot analysis, equal amounts of protein were subjected to electrophoresis in 12% SDS-PAGE gels, transferred to a PVDF membrane and blocked with TBS 1× supplemented with 0·1% Tween 20/5% non-fat dry milk. The PVDF membrane was incubated overnight at 4°C with a rabbit RGS1 antibody (rabbit, polyclonal; Pierce) at a 1:1000 dilution, washed and incubated with a secondary HRP-conjugated anti-rabbit antibody (goat; R&D Systems) at a 1:3000 dilution. After enhanced chemoluminescence reaction (SuperSignal West Femto system; Pierce), the bands were visualized. (b) All densitometric analysed data were normalized to α-tubulin and expressed as the mean ± standard deviation (SD). Comparison of each sample was performed using paired Student's t-test. Significant effects are indicated by asterisk (**P < 0·01).

cei0182-0162-sd2.tif (271.1KB, tif)

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