SEARCH

SEARCH BY CITATION

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Central nervous system (CNS)-autoreactive immune responses can exert neuroprotective effects, possibly mediated via the release of neurotrophic factors from infiltrating leucocytes. Herein, we analysed neurotrophin and cytokine mRNA levels using TaqMan polymerase chain reaction in unstimulated peripheral blood mononuclear cells (PBMCs) from multiple sclerosis (MS) patients in remission and controls. We demonstrate that mRNA for brain-derived neurotrophic factor (BDNF), but not neurotrophin-3 or nerve growth factor (NGF), is readily detectable in PBMC and that levels in MS are increased by approximately 60% compared with patients with other neurological diseases or healthy subjects. These results provide additional evidence that a potentially neuroprotective facet of autoimmune inflammation is present in MS.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Multiple sclerosis (MS) is a chronic disorder of the central nervous system (CNS), classically characterized by a relapsing–remitting disease course that in the long run leads to considerable neurological disability amongst a large proportion of the patients [1]. Strong evidence now supports the notion that MS is an autoimmune disorder in which mainly myelin-autoreactive T cells, and possibly also B cells, mount an inflammatory attack against the myelin sheaths in the CNS [1]. Traditionally, MS has mainly been considered a disease of the white matter, with relative sparing of nerve cells and axons. However, extensive axonal damage in and around inflammatory plaques has been demonstrated, and the degree of nerve cell damage may in fact be the major correlate for permanent clinical disability [2–5]. Interestingly, autoimmune reactions in the CNS may also convey protective effects in severed nerve cells. Thus, the transfer of myelin antigen-autoreactive T-cell lines increases the survival of retinal ganglion cells after optic nerve crush, and active immunization with an encephalitogenic myelin peptide exerts a similar effect on spinal motoneurons after ventral root avulsion [6, 7].

Several lines of evidence support the notion that leucocyte-mediated delivery of factors with neuronal survival-promoting activities could be the explanation for this somewhat paradoxical effect. Firstly, infiltrating immune cells in human MS plaques display immunoreactivity for brain-derived neurotrophic factor (BDNF), and both BDNF and neurotrophin-3 (NT-3) mRNA are expressed in human T-cell clones obtained from peripheral blood [8–10]. Secondly, concurrent with the survival-promoting effect on avulsed motoneurons after experimental autoimmune encephalomyelitis (EAE) immunization, infiltrating leucocytes express several neurotrophic factors, leading to highly elevated tissue levels of these factors [7].

Finally, administration of neurotrophic factors to motoneurons in culture blocks the deleterious effects of tumour necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) on these cells [7]. It is therefore conceivable that the expression of neurotrophic factors in immune cells may be of great relevance for the protection of neurons during inflammation in the CNS. However, knowledge about the regulation of NTs in the human immune system and in diseases such as MS is very scarce to date. In one study, nerve growth factor (NGF) was increased in cerebrospinal fluid of MS patients, albeit only during acute attacks [11].

We have herein examined the expression of NT mRNAs in unstimulated peripheral blood mononuclear cell (PBMC) of relapsing–remitting MS patients in remission, using a semiquantitative TaqMan real-time polymerase chain reaction (PCR) protocol. Our results demonstrate the increased expression of BDNF mRNA in MS patients compared with controls, which is consistent with an active role of this factor, perhaps as a compensatory, restorative or immunomodulatory mediator in this autoimmune neuroinflammatory disease.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Patients and control subjects Twenty patients (six men and 14 women) suffering from clinically definite MS according to the Poser criteria [12] were included in the study. Their mean age was 42 years (standard deviation (SD) = 13) and mean duration of disease was 7 years (SD = 10). All patients were ambulatory with a Kurtzke expanded disability status scale (EDSS) score <5 and displayed a relapsing–remitting disease course with a mean EDSS of 2.0 (SD = 1.0). The control populations consisted of healthy controls (HC) and patients with other neurological diseases (OND). The HC group was represented by 14 volunteers (six men and eight women) who were selected from the hospital staff. Their mean age was 38 (SD = 9). Sixteen patients (nine men and seven women) with a mean age of 50 (SD = 13) were included in the OND group. These patients could be characterized as follows: post-polio syndrome (6), disc hernia/nerve root syndrome (3), vascular disease/minor stroke (1), sudden deafness (1), polyneuropathy (1), spinal stenosis (1), myelitis (1), communicating hydrocephalus (1) and Addison's disease (1). The collection of samples for research purposes was permitted by the ethical committee of the Karolinska Hospital. Patients admitted to the outpatient clinic for an investigation of possible neuroinflammatory disease comprised the majority of both MS and OND groups. None of the patients had received treatment with corticosteroids, IFN-β, glatiramer acetate or cytostatics within 1 year before sampling. Blood was collected in heparinized glass tubes, and PBMCs were immediately isolated by density gradient centrifugation using lymphoprep (Nycomed, Oslo, Norway). Cells were collected from the interphase and washed three times with phosphate-buffered saline (PBS). No stimulation of isolated cells was performed. Cell pellets were stored at −20 °C until use.

Semiquantitative TaqMan reverse transcriptase-PCR Cell pellets were lysed, and total RNA was extracted (Qiagen total RNA extraction kit, Qiagen, Hilden, Germany). Samples were incubated with 27 kU of DNase for 30 min at 37 °C in order to avoid amplification/detection of contaminating genomic DNA. Reverse transcription was performed with 10 µl of total RNA, random hexamer primers (0.1 µg; Gibco BRL, Life Technologies, Täby, Sweden), and superscript reverse transcriptase (200 U; Gibco BRL). Amplification was performed using an ABI PRISM 7700 sequence detection system (Perkin Elmer, Norwalk, CT, USA) using the 5′ nuclease method (TaqMan) with a two-step PCR protocol (95 °C for 10 min, followed by 40 cycles of 95 °C for 15 s and 60 °C for 1 min). All primers and probes were designed with the primer express software (Perkin Elmer; Table 1), except for 18S rRNA (Perkin Elmer). In preliminary experiments, the primer pairs had been tested using a conventional PCR protocol. The PCR products were run in an agarose gel and were in all cases confined to a single band of the expected size. Sequencing of the different bands (Cybergene AB, Huddinge, Sweden) confirmed homology with the reported sequences for human BDNF, NT-3, NGF, TNF-α and interleukin-10 (IL-10), respectively. Semiquantitative assessment of mRNA levels was performed using the standard curve method, with amplification of mRNA and 18S rRNA in separate tubes (described in detail in user bulletin, No. 2, Applied Biosystems, Foster City, CA, USA 1997).

Table 1. 
Gene Oligonucleotide sequence
  1. BDNF, brain-derived neurotrophic factor; IL-10, interleukin-10; NT-3, neurotrophin-3; NGF, nerve growth factor; TNF-, tumour necrosis factor-.

BDNFForward5′-GCT GAC ACT TTC GAA CAC ATG A
 Reverse5′-CTG GAC GTG TAC AAG TCT GCG T
 Probe5′-CTG TTG GAT GAG GAC CAG AAA GTT CGG C
NT-3Forward5′-GAT AAA CAC TGG AAC TCT CAG TGC AA
 Reverse5′-GCC AGC CCA CGA GTT TAT TGT
 Probe5′-CAA ACC TAC GTC CGA GCA CTG ACT TCA GA
NGFForward5′-GTG CTG CCC CCT TCA ACA
 Reverse5′-GAA TTC GCC CCT GTG GAA
 Probe5′-TCA CAG GAG CAA GCG GTC ATC ATC C
TNF-αForward5′-CCA GGG ACC TCT CTC TAA TCA GC
 Reverse5′-CTC AGC TTG AGG GTT TGC TAC A
 Probe5′-CTC TGG CCC AGG CAG TCA GAT CAT CTT
IL-10Forward5′-CGG CGC TGT CAT CGA TTT
 Reverse5′-TTA AAG GCA TTC TTC ACC TGC TC
 Probe5′-CCC TGT GAA AAC AAG AGC AAG GCC G

Fluorescence-activated cell sorter sorting Samples for fluorescence-activated cell sorter (FACS) sorting of PBMCs were collected from two MS patients and one patient with amyotrophic lateral sclerosis (ALS). PBMCs were isolated as described above. Monoclonal antibodies were purchased from BD Biosciences (San Jose, CA, USA) and titred to optimal concentrations.

Cell suspensions were labelled with the following antihuman monoclonal antibodies in different combinations; Cy-chrome-conjugated anti-CD3, fluorescein isothiocyanate-conjugated anti-CD4 and phycoerythrin-conjugated anti-CD8, -CD14 or -CD19. The Simultest CD3/CD16+CD56 was used for the detection of natural killer (NK) cells. The samples were incubated in the dark at room temperature for 20 min, washed twice with PBS, supplemented with 0.09% NaN3 and 3% fetal calf serum and resuspended in 300 µl of PBS before analysis using a FACSVantage SE system (Becton Dickinson, San Jose, CA, USA). One region (R1) was defined in the light scatter plot corresponding to live cells, and another region (R2) delineated cells positive for the respective antibody. Cell sorting was finally performed with the sorting gate set as R1 × R2. Approximately, 1–2 × 105 cells of each subpopulation were sorted into 2 ml Eppendorf tubes. After centrifugation, the cell pellets were kept at −20 °C until RNA extraction. Sorting purity was determined to be more than 95%.

Statistical analysis Significance levels were determined using the nonparametric multiple comparison Kruskal–Wallis test and Dunn's post-hoc test. Correlation was analysed with the Spearman's rank test. All analyses were performed using graphpad prism 3.0 (Graph Pad Software, San Diego, CA, USA).

Results

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

Increased BDNF mRNA expression in PBMCs from MS patients

We analysed a relatively homogenous group of outpatients. Importantly, no patients were sampled within 4 weeks of an active attack, thus reflecting baseline levels in MS patients. All samples displayed detectable levels of BDNF, even though no PBMCs were sampled. Although the TaqMan methodology used here only gives information about relative differences in the levels of a studied transcript, the number of cycles needed before the levels of amplified product reaches detectable levels gives some indications about the abundance of starting material. The threshold cycle (Ct) values for BDNF were 27–33, suggesting a moderately abundant expression and were in the same range as those for IL-10 and TNF-α mRNAs. In contrast, NGF and NT-3 mRNAs were only detected in occasional samples and always with a Ct value >35. BDNF levels in PBMCs from healthy subjects displayed relatively little variation, whereas variation in the MS and OND groups were more pronounced, with some samples lying considerably over the median (Fig. 1). Median levels of BDNF mRNAs in the MS group were in the order of 60% higher than that of the HC and OND groups. The expression of IL-10 and TNF-α did not statistically differ between the groups. However, in a similar study consisting of more than twice as many subjects, IL-10, but not TNF-α, is significantly increased in PBMCs of MS patients compared with OND and HC groups (Khademi and Gielen, in preparation). This may suggest that the sample size analysed here was too small to detect this difference.

image

Figure 1. Expression of brain-derived neurotrophic factor (BDNF) (A), tumour necrosis factor-α (TNF-α) (B), interleukin-10 (IL-10) (C) mRNAs as a relative ratio to 18S rRNA in peripheral blood mononuclear cells (PBMCs) from patients with multiple sclerosis (MS), other neurological diseases (OND), and healthy controls (HC). Values are normalized to the HC group. (A) BDNF expression was increased in MS patients as compared with the OND (P < 0.001) and HC (P < 0.01) groups. (B and C) Expression of TNF-α and IL-10 mRNAs did not differ significantly between any of the groups (P > 0.05).

Download figure to PowerPoint

The levels of BDNF and TNF-α mRNAs displayed a low degree of correlation in MS patients (r = 0.50, 95% CI = 0.06–0.78, P < 0.05), but not in the OND and HC groups. No correlation between BDNF and IL-10 was evident in any of the groups.

BDNF mRNA is expressed in several leucocyte cell populations

The levels of NT mRNAs were also determined in FACS-sorted, unstimulated PBMCs in order to establish which cell population expressed these factors (Fig. 2). In concordance with the previously obtained results, detection of NGF and NT-3 mRNAs was only recorded in occasional samples and with high Ct values, suggesting low levels of expression. BDNF mRNA was detected in both CD4+ and CD8+ T cells, NK cells, NK T cells and B cells, but not in monocytes. Relative levels were similar in the different cell populations, but in all the three cases, the CD4+ T cells displayed approximately twice as high levels as the other cell populations.

image

Figure 2. Expression of brain-derived neurotrophic factor (BDNF) (A) and interferon-γ (IFN-γ) (B) mRNAs in fluorescence-activated cell sorter-sorted lymphocyte populations from two MS patients (MS1 and MS2) and one patient with amyotrophic lateral sclerosis (ALS). Values are expressed as a relative ratio to 18S rRNA and normalized to the mean of all values. The following leucocyte populations were sorted – CD4 T cells (CD4), CD8 T cells (CD8), natural killer cells (NK), NK T cells (NK T), CD14-positive monocytes (CD14), CD19-positive B cells (CD19). BDNF is expressed in all leucocyte populations except monocytes, whereas IFN-γ mRNA is detectable in all populations except monocytes and B cells.

Download figure to PowerPoint

The number of samples is not sufficiently large to draw conclusions about quantitative differences. However, expression patterns do not suggest any differences with regard to qualitative aspects and probable sources of BDNF mRNA in PBMCs between the two MS patients and the ALS patient.

Discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

One of the hallmarks of MS pathology is the accumulation of inflammatory cell infiltrates in the CNS parenchyma, and it is now widely accepted that leucocytes and, in particular, CD4+ T cells orchestrate the inflammatory assault directed to myelin sheaths. The notion that inflammation also has deleterious consequences for nerve cells is supported by studies reporting the highest degree of axonal damage during the acute formation of plaques [13, 14], as well as by studies reporting acute neuronal loss after monophasic EAE [15]. However, the role leucocytes may play in nerve cell damage that accompanies white matter destruction remains unclear.

Activated T cells infiltrating the CNS during inflammatory relapses in MS or EAE are known to express high levels of the two proinflammatory cytokines IFN-γ and TNF-α[16], and many studies provide evidence that link these substances with processes that are known to be harmful to neurons, for example induction of glutamate agonists [17, 18] and nitric oxide [19, 20]. There are also data suggesting that proinflammatory cytokines could have direct cell-toxic effects on neurons [7]. Nonetheless, accumulating experimental data in the past few years have paved the ground for a new concept in autoimmunity, namely that autoimmune reactions involving T cells can be neuroprotective [21]. Even if there may exist additional ways in which CNS-infiltrating immune cells confer protection for neurons, the most attractive hypothesis proposed to date is that this is mediated via the release of neurotrophic substances [7, 9, 10]. BDNF belongs to the NT family of neurotrophic factors that are known to be of great importance for the regulation of neuronal survival during development, but they also reduce neuronal loss or atrophy after axonal lesions in the adult [22–24].

Our data demonstrate increased levels of BDNF in PBMCs of MS patients, a finding that provides additional evidence that a potentially neuroprotective aspect of autoimmune inflammation is indeed present in human neuroinflammatory disease. Inter-individual differences in the levels of BDNF were large in the MS group. This finding may reflect the biology of the disease, with a relapsing–remitting nature and a notoriously variable disease course.

Our analysis of expression in restricted leucocyte populations failed to detect BDNF mRNA in monocytes, which is interesting in light of a recent report demonstrating that the majority of BDNF-immunopositive cells in CNS autopsy material from MS patients expressed macrophage markers [10]. Taken together, this suggests that upregulation of BDNF expression may occur, as monocytes recruited from the blood differentiate into tissue macrophages in the nervous system.

Another important aspect of the presence of BDNF in leucocytes is its potential role in immune regulation, as there is now experimental data that ascribe a direct regulatory influence of neurotrophic factors on the inflammation itself. Thus, intrathecal delivery of NGF abrogates EAE in marmosets and was associated with a shift in the expression of cytokines, from IFN-γ to IL-10 [25]. T cells genetically engineered to express NGF ameliorate EAE in the rat and inhibit transendothelial migration of monocytes in vitro[26]. The observed BDNF expression in leucocytes of MS patients might therefore indicate that BDNF has an immunoregulatory function.

In conclusion, we here demonstrate moderately elevated levels of BDNF in relapsing–remitting MS patients in remission compared with controls and the expression of this NT in several leucocyte subsets. These findings support the notion that leucocyte-mediated delivery of neurotrophic substances may be of relevance in human neuroinflammatory disease.

Further studies into ways by which leucocytes mediate neuroprotection as well as the immunomodulatory effects of neurotrophic factors can lead to new treatment strategies in neurodegenerative and neuroinflammatory diseases.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References

We thank Associate Prof. Robert A. Harris for expert advice. This work was supported by Åke Wibergs, Magn Bergvalls and Tore Nilsons stiftelse, NHR and the Swedish Medical Research Council.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results
  6. Discussion
  7. Acknowledgments
  8. References
  • 1
    Steinman L. Multiple sclerosis: a coordinated immunological attack against myelin in the central nervous system. Cell 1996;85: 299302.
  • 2
    Davie CA, Barker GJ, Webb S et al. Persistent functional deficit in multiple sclerosis and autosomal dominant cerebellar ataxia is associated with axon loss. Brain 1995;118: 158392.
  • 3
    De Stefano N, Matthews PM, Fu L et al. Axonal damage correlates with disability in patients with relapsing-remitting multiple sclerosis. Results of a longitudinal magnetic resonance spectroscopy study. Brain 1998;121: 146977.
  • 4
    Lee MA, Blamire AM, Pendlebury S et al. Axonal injury or loss in the internal capsule and motor impairment in multiple sclerosis. Arch Neurol 2000;57: 6570.
  • 5
    Reddy H, Narayanan S, Matthews PM et al. Relating axonal injury to functional recovery in MS. Neurology 2000;54: 2369.
  • 6
    Moalem G, Leibowitz-Amit R, Yoles E, Mor F, Cohen I, Schwartz M. Autoimmune T cells protect neurons from secondary degeneration after central nervous system axotomy. Nat Med 1999;5: 4955.
  • 7
    Hammarberg H, Lidman O, Lundberg C et al. Neuroprotection by encephalomyelitis: rescue of mechanically injured neurons and neurotrophin production by CNS-infiltrating T and natural killer cells. J Neurosci 2000;20: 528391.
  • 8
    Besser M, Wank R. Cutting edge: clonally restricted production of the neurotrophins brain-derived neurotrophic factor and neurotrophin-3 mRNA by human immune cells and Th1/Th2-polarized expression of their receptors. J Immunol 1999;162: 63036.
  • 9
    Kerschensteiner M, Gallmeier E, Behrens L et al. Activated human T cells, B cells, and monocytes produce brain-derived neurotrophic factor in vitro and in inflammatory brain lesions: a neuroprotective role of inflammation? J Exp Med 1999;189: 86570.
  • 10
    Stadelmann C, Kerschensteiner M, Misgeld T, Bruck W, Hohlfeld R, Lassmann H. BDNF and gp145trkB in multiple sclerosis brain lesions: neuroprotective interactions between immune and neuronal cells? Brain 2002;125: 7585.
  • 11
    Laudiero LB, Aloe L, Levi-Montalcini R et al. Multiple sclerosis patients express increased levels of beta-nerve growth factor in cerebrospinal fluid. Neurosci Lett 1992;147: 912.
  • 12
    Poser CM, Paty DW, Scheinberg L et al. New diagnostic criteria for multiple sclerosis: guidelines for research protocols. Ann Neurol 1983;13: 22731.
  • 13
    Ferguson B, Matyszak MK, Esiri MM, Perry VH. Axonal damage in acute multiple sclerosis lesions. Brain 1997;120: 3939.
  • 14
    Trapp B, Peterson J, Ransohoff R, Rudick R, Mørk S, Bø L. Axonal transection in the lesions of multiple sclerosis. N Engl J Med 1998;338: 27885.
  • 15
    Smith T, Groom A, Zhu B, Turski L. Autoimmune encephalomyelitis ameliorated by AMPA antagonists. Nat Med 2000;6: 626.
  • 16
    Olsson T. Critical influences of the cytokine orchestration on the outcome of myelin antigen-specific T-cell autoimmunity in experimental autoimmune encephalomyelitis and multiple sclerosis. Immunol Rev 1995;144: 24568.
  • 17
    Piani D, Spranger M, Frei K, Schaffner A, Fontana A. Macrophage-induced cytotoxicity of N-methyl-d-aspartate receptor positive neurons involves excitatory amino acids rather than reactive oxygen intermediates and cytokines. Eur J Immunol 1992: 22;242936.
  • 18
    Giulian D, Corpuz M, Chapman S, Mansouri M, Robertson C. Reactive mononuclear phagocytes release neurotoxins after ischemic and traumatic injury to the central nervous system. J Neurosci Res 1993;36: 68193.
  • 19
    Chao CC, Hu S, Ehrlich L, Peterson PK. Interleukin-1 and tumor necrosis factor-alpha synergistically mediate neurotoxicity: involvement of nitric oxide and of N-methyl-d-aspartate receptors. Brain Behav Immun 1995;9: 35565.
  • 20
    Merrill JE, Ignarro LJ, Sherman MP, Melinek J, Lane TE. Microglial cell cytotoxicity of oligodendrocytes is mediated through nitric oxide. J Immunol 1993;151: 213241.
  • 21
    Schwartz M, Kipnis J. Protective autoimmunity: regulation and prospects for vaccination after brain and spinal cord injuries. Trends Mol Med 2001;7: 2528.
  • 22
    Kishino A, Ishige Y, Tatsuno T, Nakayama C, Noguchi H. BDNF prevents and reverses adult rat motor neuron degeneration and induces axonal outgrowth. Exp Neurol 1997;144: 27386.
  • 23
    Kobayashi NR, Fan DP, Giehl KM, Bedard AM, Wiegand SJ, Tetzlaff W. BDNF and NT-4/5 prevent atrophy of rat rubrospinal neurons after cervical axotomy, stimulate GAP-43 and Talpha1-tubulin mRNA expression, and promote axonal regeneration. J Neurosci 1997;17: 958395.
  • 24
    Hammond EN, Tetzlaff W, Mestres P, Giehl KM. BDNF, but not NT-3, promotes long-term survival of axotomized adult rat corticospinal neurons in vivo. Neuroreport 1999;10: 26715.
  • 25
    Villoslada P, Hauser SL, Bartke I et al. Human nerve growth factor protects common marmosets against autoimmune encephalomyelitis by switching the balance of T helper cell type 1 and 2 cytokines within the central nervous system. J Exp Med 2000;191: 1799806.
  • 26
    Flugel A, Matsumuro K, Neumann H et al. Anti-inflammatory activity of nerve growth factor in experimental autoimmune encephalomyelitis: inhibition of monocyte transendothelial migration. Eur J Immunol 2001;31: 1122.