Neurotrophic Factors and Clinical Recovery in ... - Wiley Online Library

doi: 10.1111/j.1365-3083.2005.01649.x ............................................................................................................................................................................................................

Neurotrophic Factors and Clinical Recovery in Relapsing-Remitting Multiple Sclerosis M. Caggiula, A. P. Batocchi, G. Frisullo, F. Angelucci, A. K. Patanella, C. Sancricca, V. Nociti, P. A. Tonali & M. Mirabella

Abstract Department of Neuroscience, Institute of Neurology, Catholic University School of Medicine, Rome, Italy. Received 18 February 2005; Accepted in revised form 1 June 2005 Correspondence to: Dr M. Mirabella, MD, PhD, Department of Neuroscience, Institute of Neurology, Catholic University School of Medicine, Largo Gemelli 8, 00168 Rome, Italy. E-mail: [email protected]

Pathogenic autoimmune cells are demonstrated to be able to produce neurotrophic factors during acute phase of multiple sclerosis (MS). In this study, we determined the production of various neurotrophins [brain-derived neurotrophic factor (BDNF), nerve growth factor (NGF), glial cell line-derived neurotrophic factor (GDNF), neurotrophin 3 (NT3) and neurotrophin 4 (NT4)] and some pro-inflammatory cytokines [tumour necrosis factor-a (TNF-a) and interferon-g (IFN-g)] by unstimulated peripheral blood mononuclear cells (PBMC) in 21 relapsing-remitting MS patients during different phases of disease (stable, relapse and post-relapse). During acute phase of disease, we detected a considerable increase of BDNF, TNF-a and IFN-g production, while significantly higher levels of GDNF, NGF, NT3 and NT4 were found in post-relapse phase. When neurotrophin production was correlated with clinical outcome (complete or partial recovery from new symptoms), we found a significantly higher BDNF production in relapse phase followed by increased GDNF, NGF, NT3 and NT4 levels during post-relapse phase in subjects with complete remission only. During relapse phase, we detected a significant increase of pro-inflammatory cytokines, that was more evident in patients with partial recovery. The neuroprotective potential of immune cells seems to be inversely correlated with disease duration and with the age of patients.

Introduction Multiple sclerosis (MS) is a chronic disease of the central nervous system (CNS) characterized by the presence, within the brain and spinal cord white matter, of inflammatory infiltrates containing few autoreactive T cells and a multitude of pathogenic non-specific mononuclear cells in areas of demyelination, axonal loss and severe glial scarring. Inflammatory mechanisms contribute to axonal pathology as well as to demyelination, [1] but autoimmune cells were also demonstrated to have neuroprotective properties, at least partly mediated by the release of neurotrophic factors [2, 3]. Neurotrophins are a group of structurally related proteins including three families of growth factors: nerve growth factor (NGF) family [brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT3), neurotrophin 4/5 (NT4/5)], glial cell line-derived neurotrophic factor (GDNG) family ligands (GDNF, neurturin, artemin and persephin) and neuropoietic cytokines [ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF)] [4].

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Such protein factors are able to prevent neural death, to favour the recovery process, neural regeneration and remyelination [4]. BDNF has a role in promoting the survival and differentiation of neurons; this neurotrophin is one of the most potent factors supporting neuronal survival and regulating neurotransmitter release and dendritic growth [5]. Several studies have shown that the therapeutic application of BDNF prevents neuronal degeneration after experimental axotomy and other forms of neuronal injury [6]. GDNF promotes axonal growth after partial and complete spinal cord transections and induces remyelination [7]. NGF has been demonstrated to promote the biosynthesis of myelin by oligodendrocytes in CNS and by Schwann cells in the peripheral nervous system [8, 9] as well as the differentiation of oligodendrocytes by cells of the subventricular zone in experimental autoimmune encephalomyelitis (EAE) [10]. Moreover, the intracerebroventricular administration of NGF can delay the onset of EAE [11].

# 2005 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 62, 176–182

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Most members of the neurotrophin family (BDNF, NGF, GDNF, NT3, NT4) can be produced and secreted within the immune system (by B and T lymphocytes and monocytes) [4], and antigen activation significantly increases NT secretion by lymphocytes [12]. Immune cells express neurotrophin receptors and seem to be the target of neurotrophin autocrine and paracrine actions in turn [4, 12, 13]. Increased neurotrophin production has been shown in inflammatory infiltrates during EAE: NGF was expressed mainly in macrophages [14], while very high levels of BDNF, NT-3 and GDNF mRNAs were present in T and NK cell populations infiltrating the CNS [15]. In MS patients, a significant number of immune cells containing BDNF was detected in actively demyelinating areas of MS lesions [5], and an increase of NGF and CNTF levels in cerebrospinal fluid (CSF) was demonstrated during relapse and recovery phases, respectively [16, 17]. BDNF production by peripheral blood mononuclear cells (PBMC) in MS patients is higher during relapse and in the recovery phase, as compared with values detected in the stable phase of the disease [18]. The aims of this study were: (i) to determine neurotrophin (BDNF, NGF, GDNF, NT3 and NT4) and proinflammatory cytokine [tumour necrosis factor-a (TNF-a) and interferon-g (IFN-g)] production by PBMCs in relapsing-remitting MS (RRMS) patients at different phases of disease (stable, relapse and postrelapse) and (ii) to evaluate whether clinical recovery is correlated with different neurotrophic factor levels.

Materials and methods Patients. We serially studied 21 consecutive patients with definite MS (16 females and five males) [19]; all subjects presented a RR course of disease and they had never been treated with any immunomodulatory drugs except corticosteroids. Their mean age was 27.6 years and mean disease duration was 40.7 months. Clinical data and blood samples were obtained in stable phase of disease, during relapse and in post-relapse time. The Expanded Disability Status Scale (EDSS) was used to score degree of disability. The clinical examination of all patients at different times was performed by the same neurologist. The clinical and haematological control in stable phase was performed at least 3 months after the last relapse or steroid administration. The relapse phase occurred 86 (37) days after control in stable phase. We considered a ‘relapse’ as the occurrence of new neurological symptoms with progression in disability (increase of EDSS score >0.5) lasting for at least 24 h, associated with detection of new gadolinium-enhancing lesions on brain and spinal cord magnetic resonance imaging (MRI) examination. All patients underwent MRI and were treated with

intravenous methilprednisolone (20 mg/kg/die for 5 days) at time of relapse. Blood was drawn before starting therapy in order to avoid corticosteroid-induced variations in neurotrophin production [20]. The post-relapse control was performed within 3 months (63 32 days) from relapse. At the end of the follow-up, patients were divided into two groups based on the full or partial recovery from new neurological symptoms in post-relapse phase. All patients gave their informed consent to participate in the study, which was approved by the Ethical Committee of our Institution. Separation of PBMCs and cell culture. PBMCs were isolated from venous blood by density gradient centrifugation (7860 g, 30 min) over a Ficoll-Hypaque density gradient (Pharmacia, Uppsala, Sweden). PBMCs were then harvested by pipetting cells from the Ficoll/serum interface and washed twice. Cells were cultured for 24 h in 24-well plates at a density of 5 106/ml in RPMI 1640 (EuroClone, West York, UK) containing 2 nM L-glutamine and 5% fetal calf serum. At the end of incubation, cell-free supernatants were harvested and stored at 80 C until assayed. Measurement of cytokine and neurotrophin production by PBMCs. Spontaneous cytokine (TNF-a and IFN-g) and neurotrophin (BDNF, NGF, GDNF, NT3 and NT4) production was measured by enzyme-linked immunoabsorbent assay using commercial kits (R&D Systems, MN, USA) following manufacturer’s instructions. All assays were performed on F-bottom 96-well plates (Nunc, Wiesbaden, Germany). Tertiary antibodies were conjugated to horseradish peroxidase. Wells were developed with tetramethylbenzidine and measured at 450/570 nm. Cytokine and neurotrophin concentrations were determined from the regression line for a standard curve generated by using highly purified recombinant human cytokine or neurotrophin at various concentrations performed contemporaneously with each assay. The standard curve also served as an internal control over the sensitivity and range of each assay. All samples were assayed in duplicate, and quality control pools at low, normal and high concentrations were present in each assay. Data were expressed as pg/ml. There was no cross-reactivity or interference between related neurotrophins (BDNF, NGF, GDNF, NT3 and NT4). MRI. All patients underwent MRI at relapse time. MRI data were acquired on high-resolution 1.5 Tesla system with 5-mm slice thickness. Scanning sessions included proton density [echo time (TE) 20/repetition time (TR) 2500], T2-weighted (TE 80/TR 2500) and T1-weighted (TE 17/TR 600) images. The T1-weighted images were acquired before and 10 min after IV injection of gadolinium-diethylenetriaminepentaacetic acid (0.1 mmol/kg). Statistical analysis. Variations of cytokine (TNF-a and IFN-g) and neurotrophic factor (BDNF, GDNF, NGF,


178 Neurotrophins and Clinical Recovery in MS M. Caggiula et al. ............................................................................................................................................................................................................ NT3 and NT4) production by PBMCs at different phases of disease (stable, relapse and post-relapse phases) were assessed across all subjects and separately for the two groups of patients by ANOVA. When significant differences were obtained, post hoc comparisons were performed using Fisher’s PLSD test. A P value 0.5) as compared with the prerelapse phase. The two groups of patients were comparable as to sex, relapse rate in the past 2 years and baseline EDSS, but significantly differed for disease duration (Table 1), with a longer history of disease in subjects who only obtained partial recovery in post-relapse phase as compared with patients with complete remission. Patients who only showed a partial recovery were also characterized by a relatively higher age than the ones with complete remission, although this difference was not statistically significant. There were no differences between the groups in the average time span from baseline control to relapse and from relapse to post-relapse examination (mean time to relapse in patients with complete recovery from new neurological symptoms 81 days, in patients with partial recovery 85 days; mean time from relapse in patients with complete recovery from new neurological symptoms 63 days and in patients with partial recovery 66 days). There was no difference in the number of enhancing lesions between groups (Table 1). In stable phase of disease, levels of all neurotrophic factors studied – BDNF, NGF, GDNF, NT3 and NT4 – were comparable between the two groups of patients (in patients with complete recovery from new neurological symptoms, baseline BDNF min–max levels 83–779 pg/ ml, NGF 0–20 pg/ml, GDNF 0–3 pg/ml, NT3 2–20 pg/ ml, NT4 1–5 pg/ml; in patients with partial recovery, baseline BDNF min–max levels 126–250 pg/ml, NGF 0– 11 pg/ml, GDNF 0–2 pg/ml, NT3 2–8 pg/ml, NT4 1–5 pg/ml). At that time, GDNF, NGF, NT3 and NT4 concentrations were very low in all samples. Moreover, pro-inflammatory cytokine levels, TNF-a and IFN-g were comparable between the groups (in patients with complete recovery from new neurological symptoms, baseline TNF-a min–max levels 43–110 pg/ml, IFN-g 10–65 pg/ml; in patients with partial recovery, baseline TNF-a min–max levels 10–36 pg/ml, IFN-g 5–36 pg/ml). The increase of TNF-a (P < 0.05) and IFN-g during relapse was evident in both groups of patients, but it was higher (although not statistically significant) in patients who obtained partial recovery from new symptoms than in patients with full recovery (Fig. 2A,B) (in patients with complete recovery from new neurological symptoms, baseline TNF-a min–max levels 43–110 pg/ml, relapse TNF-a min–max levels 150–700 pg/ml; baseline IFN-g min–max levels 10–65 pg/ml, relapse IFN-g min–max levels 30– 310 pg/ml; in patients with partial recovery, baseline TNF-a min–max levels 10–36 pg/ml, relapse TNF-a min–max levels 100–1500 pg/ml; baseline IFN-g min–max levels 5–36 pg/ml, relapse IFN-g min–max levels 30–405 pg/ml). IFN-g elevation also persisted in


M. Caggiula et al. Neurotrophins and Clinical Recovery in MS 179 ............................................................................................................................................................................................................ Table 1 Clinical characteristics of the 21 relapsing-remitting multiple sclerosis (RRMS) patients studied

Number of patients Females/males Age (years, mean SD) Disease duration (months, mean SD) Number of relapses and CS treatment (in previous 2 years) Expanded disability status scale (EDSS) at stable phase (range) EDSS at acute phase (range) Gadolinium-enhancing lesions at relapse time

Total

With complete recovery

With partial recovery

P value

21 16/5 27.6 8.8 40.7 31.6 2 0.9 0–3.5 1.5–4 1.3 0.6

15 13/2 26.6 29.8 2.1 0–3 1.5–4 1.3

6 3/3 30.2 9.3 68 46 1.8 0.7 0–3.5 2–4 1.2 0.4

NS NS NS NS NS NS

post-relapse phase in the former group (Fig. 2B) (in patients with complete recovery from new neurological symptoms, post-relapse TNF-a min–max levels 20– 100 pg/ml; post-relapse IFN-g min–max levels 10–60 pg/ ml; in patients with partial recovery, post-relapse TNF-a min–max levels 15–95 pg/ml; post-relapse IFN-g min– max levels 30–180 pg/ml). Statistical analysis showed a significant correlation of clinical outcome with BDNF production (Fig. 2C). Levels of BDNF in the supernatants of unstimulated PBMCs from patients with complete remission of symptoms after corticosteroid therapy were significantly higher than values detected in patients with partial remission (P < 0.01) (Fig. 2C). During relapses, we found a significant increase of BDNF level in supernatants of PBMCs as compared with concentrations obtained during stable phases of disease only in the group of patients with complete recovery (P < 0.01) (Fig. 2C) (in patients with complete recovery from new neurological symptoms relapse BDNF min–max levels 312–1500 pg/ml; in patients with partial recovery relapse BDNF min–max levels 94–393 pg/ml). At that time, no significant variations were found in BDNF values detected in patients with partial recovery. There was no main variation of the other neurotrophic factors studied (NGF, GDNF, NT3 and NT4) in the two groups during acute phase (Fig. 2D–G). On post-relapse control, an increment of BDNF production persisted in the group of patients who obtained full recovery from new neurological symptoms after corticosteroid therapy (in patients with complete recovery from new neurological symptoms, post-relapse BDNF min–max levels 102–1200 pg/ml; in patients with partial recovery, post-relapse BDNF min–max levels 100–342 pg/ml) and an increase of NGF (P < 0.05), GDNF (P < 0.05), NT3 (P < 0.05) and NT4 production was observed in this group (Fig. 2D–G) (post-relapse NGF min–max levels 5–40 pg/ml, GDNF 2–12 pg/ml, NT3 2–45 pg/ml and NT4 2–40 pg/ml). At that time, no significant variations of all neurotrophic factors studied were found in patients with partial recovery (post-relapse NGF min–max levels 0–11 pg/ml, GDNF 1–3 pg/ml, NT3 1–3 pg/ml and NT4 1–5 pg/ml).

8.7 15 0.9

0.6

Discussion Although autoimmune responses contribute to the formation and maintenance of MS lesions [21], an increasing body of experimental evidence supports a potentially beneficial effect of inflammation [2]. That effect is, at least in part, mediated by the release of neurotrophic factors that may protect neuronal population (BDNF and NT3), enhance neuronal survival (BDNF, NGF, NT3 and NT4), promote axon regeneration and support remyelination (NGF, GDNF, NT3 and BDNF) [4, 7, 11, 12]. In actively demyelinating areas, a large quantity of BDNF is mainly produced by immune cells [22], and most neurotrophins appear to be present at the actively demyelinating edge of lesions, where they protect axons at high risk of bystander damage [4]. In addition to the neurotrophic effect, locally produced neurotrophins seem to have an immunomodulatory function by interacting with local microglia as well as invading immune cells. In fact, cytokines secreted in MS acute phase, such as interleukin-1 (IL-1), TNF-a, IL-4 and IL-5, stimulate NGF production by astrocytes; such neurotrophin might in turn modulate cytokine expression by inflammatory and glial cells. In EAE, NGF down-regulates IFN-g production by T cells infiltrating the CNS and` IL-10 production by glial cells in both inflammatory lesions and normal-appearing white matter [11]. Furthermore, it has been demonstrated that neurotrophins induce a down-regulation of the class II major histocompatibility complex molecules in microglial cells, also down-regulating costimulatory molecules [23, 24]. Neurotrophins can regulate the migration of monocytes across the blood–brain barrier and the release of proinflammatory cytokines [25], thereby helping to suppress the formation of new lesions. In chronic MS plaques, lower levels of endogenous neurotrophins are present than in early stages of lesion development and that may partly account for the ongoing axonal degeneration in these plaques during progressive phases of disease [4]. Previous studies demonstrated that the levels of BDNF in the supernatants of unstimulated and phytohaemagglutinin (PHA)-, anti-OKT3 Ab- and mannose-binding protein (MBP)-stimulated PBMCs from RRMS patients in a


180 Neurotrophins and Clinical Recovery in MS M. Caggiula et al. ............................................................................................................................................................................................................

A 1400

With complete recovery With partial recovery

B


250

* 200

1000 800

IFNγ (pg/ml)

TNF-α (pg/ml)

1200

*

600

150

100

400 50 200 0

0 Stable phase

C

Relapse

Post-relapse phase

Stable phase

D

With complete recovery With partial recovery 1200

**

*

17.5 15

800 NGF (pg/ml)

BDNF (pg/ml)

Post-relapse phase

With complete recovery With partial recovery 20

1000

Relapse

600 400

12.5 10 7.5 5

200

2.5

0

0 Stable phase

Post-relapse phase

Stable phase


E

14

3

12 NT3 (pg/ml)

2 1.5

Post-relapse phase

*

16

3.5

2.5

Relapse


F

*

4

GDNF (pg/ml)

Relapse

10 8 6

1

4

0.5

2

0

0 Stable phase

Relapse

Post-relapse phase

Stable phase

Relapse


G 10 9 8 NT4 (pg/ml)

7 6 5 4 3 2 1 0 Stable phase

Relapse

Post-relapse phase

stable clinical phase did not differ from the ones found in control subjects [18], whereas BDNF mRNA levels in PBMCs from RRMS patients in stable phase of disease were higher in comparison with healthy subjects [26]. During relapses, an increase of BDNF levels in the serum, CSF and in the supernatants of unstimulated and PHA-, anti-OKT3 Ab- and MBP-stimulated PBMCs was found, compared with values detected in stable phase of disease [18]. The BDNF increment in the supernatants of unstimulated and PHA-, anti-OKT3

Post-relapse phase

Figure 2 Neurotrophic factor and cytokine levels in supernatants of unstimulated peripheral blood mononuclear cells from relapsing-remitting MS (RRMS) patients at different phases of disease (stable, relapse and post-relapse phase). Data are the means SEM. Asterisks indicate statistical significances (*P < 0.05, **P < 0.01). (A) Tumour necrosis factor-a (TNF-a) levels in patients with partial (white columns) and complete (black columns) recovery from new neurological symptoms in post-relapse phase. During relapse phase, we found a significant increase of TNF-a (P < 0.05) in both groups of patients, but it was higher (although not so much as to be statistically significant) in patients who obtained partial recovery from new symptoms than in patients with full recovery. In post-relapse phase, we detected a dramatic decrease of TNF-a (P < 0.05) both in patients with total and with partial recovery from new symptoms. (B) Interferon-g (IFN-g) levels in patients with partial (white columns) and complete (black columns) recovery from new neurological symptoms in post-relapse phase. An increase of IFN-g production by peripheral blood mononuclear cell (PBMCs), as compared with baseline values, was observed in both groups of patients. IFN-g elevation also persisted in post-relapse phase in subjects who did not achieve full recovery from relapse symptoms after corticosteroid therapy. (C) Brain-derived neurotrophic factor (BDNF) levels in patients with partial (white columns) and complete (black columns) recovery from new neurological symptoms in post-relapse phase. The two groups of patients significantly differ for BDNF production (P < 0.01). The levels of BDNF detected in the supernatants of unstimulated PBMCs from patients with partial remission of symptoms after corticosteroid therapy are considerably higher than the ones found in patients with complete recovery. In the latter group, a significant increase of BDNF level was detected in supernatants of PBMCs collected during relapse as compared with concentrations obtained during stable phases of disease (P < 0.01) and an increase of BDNF production, as compared with concentrations obtained during stable phases, persisted in post-relapse phase. (D–G), glial cell line-derived neurotrophic factor (GDNF), nerve growth factor (NGF), neurotrophin 3 (NT3) and neurotrophin 4 (NT4) levels in patients with partial (white columns) and complete (black columns) recovery from new neurological symptoms in post-relapse phase. No main variation of these neurotrophic factors was found at relapse time. An increase of NGF (P < 0.05), GDNF (P < 0.05), NT3 (P < 0.05) and NT4 production was observed in the postrelapse phase as compared with concentrations obtained during stable phases only in the group of patients with complete recovery.

Ab- and MBP-stimulated PBMCs persisted during recovery phase [18]. In our study, we detected a significant increase of BDNF production in relapse phase, associated with the increase of pro-inflammatory cytokines (TNF-a and IFNg). Although the levels of NGF, GDNF, NT3 and NT4 were very low at stable and relapse phases of disease, a significant post-relapse increase of GDNF, NGF, NT3 and NT4 production was detected during post-relapse phase. However, when we considered the groups of


M. Caggiula et al. Neurotrophins and Clinical Recovery in MS 181 ............................................................................................................................................................................................................

patients separately, according to their clinical outcome, these findings were even more evident in the group of patients showing complete remission of symptoms after corticosteroid therapy and were absent in the very small group of subjects who only achieved a partial remission of new symptoms. BDNF production was strictly correlated with clinical outcome, and BDNF levels detected in patients with full recovery from relapse symptoms were significantly higher than values found in patients with partial recovery. Subjects with complete recovery from relapse symptoms in the post-relapse phase were characterized by a significant increase of BDNF during relapse. The enhanced production of BDNF persisted in that group of patients during the recovery phase associated with a considerable increase of GDNF, NGF, NT3 and NT4. By contrast, no significant variation of the neurotrophic factors studied was observed in patients with a partial recovery at any time point. Although the increase of pro-inflammatory cytokines (TNF-a and IFN-g) during relapse was evident in both groups of patients, it was higher in patients who obtained partial recovery from new symptoms than in patients with full recovery. IFN-g elevation also persisted in post-relapse phase in the former group. The two groups significantly differed as to disease duration: patients with a partial remission of relapse symptoms had a longer history of disease, compared to subjects with full recovery. Patients with partial remission were also characterized by a relatively higher age, although not statistically significant, than the ones with complete remission. No other clinical feature studied was different in the two groups. Previous studies have demonstrated that PBMCs from secondary progressive MS patients show lower BDNF production than the ones from RRMS subjects, and such reduction is more marked in the patients with progression in disability than in the ones with a stable neurological impairment [18]. Thus, it has been suggested that the reduction of neuroprotective potential of inflammatory cells may correspond to a shift toward a progressive phase of illness. In our study, patients with an incomplete recovery after relapse seem to show a reduced neuroprotective activity of inflammatory cells, revealed by a lower production of neurotrophic factors associated with a relatively higher production of pro-inflammatory cytokines. Those defective neuroprotective mechanisms seem to be correlated with a longer disease duration and a relatively higher age. Even though the number of patients is too small to allow a definite conclusion, our data show that the production of neurotrophic factors during both active inflammatory phase and post-relapse phase is well correlated with clinical recovery, while a progression in disability is associated with a reduced neuroprotective potential of inflammatory cells. The beneficial effect of inflammation seems

to be more effective in patients with a shorter disease duration. These data bear out the neuroprotective effect of inflammation in the early phases of MS and a reduced neuroprotective potential of immune cells associated with accumulation of disability. Further studies carried out on larger series of patients are needed to confirm our findings, that may have implications for the therapy, in view of the limited success of non-selective immunotherapies in MS. In fact, a better knowledge of the production balance between beneficial and detrimental factors by immune cells during natural history of MS would allow a more targeted use of immunosuppressive agents, which could be exclusively administered when the noxious aspects of inflammation prevail. Additional studies are also necessary to clarify the effect of currently available immunomodulatory drugs on the neuroprotective function of autoimmune cells.

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