Multiple sclerosis patients express increased levels of β-nerve growth factor in cerebrospinal fluid

Neuroscience Letters, 147 (1992) 9-12

9

© 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00 NSL 09077

Multiple sclerosis patients express increased levels offl-nerve growth factor in cerebrospinal fluid L. Bracci Laudiero a, L. Aloe a, R. Levi-Montalcini a, C. Buttinelli b, D. Schilter c, S. Gillessen ¢ and U. Otten ~ alnstitute of Neurobiology, CNR, Rome (Italy}, bDepartment of Neurological Sciences, Universit& "La Sapienza" Rome (Italy) and CDepartment of Physiology, University of Basel, Basel (Switzerland) (Received 27 July 1992; Revised version received 17 August 1992; Accepted 18 August 1992)

Key words: Multiple sclerosis; Cerebrospinal fluid; ,&Nerve growth factor (NGF); Inflammation We describe the measurement of r-nerve growth factor (NGF) content in cerebrospinal fluid (CSF) from multiple sclerosis (MS) patients compared with CSF from age-matched normal subjects using a specific sandwich immunoassay (ELISA). During acute attacks patients exhibit a significant increase of NGF content compared to controls. In contrast during remission the mean NGF levels in CSF markedly decrease. These results strongly indicate that increased NGF production in CSF is a characteristic feature of the MS inflammatory response.

Normal development and function of neurons depend on the presence of neurotrophins [16, 21]. The best characterized neurotrophin is r-nerve growth factor (NGF). N G F supports the survival of sympathetic neurons and neural crest-derived sensory neurons [16], as well as central cholinergic neurons [15, 30]. In addition to its neurotrophic actions N G F modulates inflammatory and immune responses [2, 17, 22-25, 27]. Human lymphocytes express functional N G F receptors [25] and N G F induces proliferation as well as the differentiation of B-cells [14, 25]. Accumulation of N G F at sites of lesion [18] and inflammation [29] suggests that inflammatory stimuli contribute to increasing N G F production. Thus N G F may act as an immunomodulator mediating cross talk between cells of the nervous and immune systems. Neurodegenerative diseases which involve inflammation may provide valuable information about the pathophysiological role of NGF-cytokine interactions. Multiple sclerosis (MS) is a chronic recurrent inflammatory disease of the central nervous system (CNS) which may be an autoimmune disorder [12]. It is characterized by a patchy destruction of the myelin sheath during which lymphocytes and monocytes infiltrate white matter and release cytokines when stimulated. Recent studies indicate that cytokines, including interleukin- 1 (IL- 1), interleukin-6 (IL-6) and tumor necrosis factor-a (TNF-~),

Correspondence: U. Otten, Department of Physiology, University of Basel, Vesalgasse 1, 4051 Basel, Switzerland.

are potent inducers of N G F synthesis both in CNS [8, 9] and in peripheral tissues [18, 26]. We have used a specific sandwich immunoassay [ELISA] to measure developmental changes in N G F content in cerebrospinal fluid (CSF) from newborn to adult humans without neuronal inflammatory disease. Subsequently we investigated variation of N G F levels with the disease process in MS patients. More than 80 human CSF samples from newborns and children were provided by the Department of Microbiology of Kinderspital Basel, and adult CSF was provided by Central Laboratories of Kantonsspital, Basel, We analysed 15 CSF samples of definite MS patients (followed at the 'Multiple Sclerosis Center' of the Department of Neurological Science, University 'La Sapienza', Rome, Italy), 7 males and 8 females, mean age 45 years, range 19-54. Twelve patients had a relapsing-remitting and 3 a chronic-progressive form of MS. The CSF was divided into 2 groups according to the clinical diagnosis (Table I) by parameters such as nuclear magnetic resonance (NMR) analysis, the IgG index (CSF IgG/plasma IgG):(CSF albumin/plasma albumin), oligoclonal IgG bands and absolute IgG values. Samples were taken (a) during acute attack (relapsing phase) defined as the occurrence of a new symptom or symptoms of neurological dysfunction lasting more than 24 h, and (b) during remission. In addition, CSFs from 8 patients (4 males and 4 females, mean age 43.3, range 21-78) with other neurological diseases (OND), (1 headache, 1 cerebellar neoplastic syndrome, 1 myelopathy, 2 polyneu-

10 TABLE I DIAGNOSTIC PARAMETERS DISTINGUISHING MS FROM OTHER NEUROLOGICAL DISORDERS (OND) IgG index is equivalent/* to (CSF IgG/plasma IgG):(CSF albumin/ plasma albumin). Values > 0.7 were taken as intrathecal IgG. NMR (nuclear magnetic resonance) imaging: 82-100% of patients with clinically defined MS show multifocal cerebral white matter lesions by NMR analysis. MS acute (n=4)

Mononuclear cells (5×106/1) Range Mean % abnormality IgG values (1.3-2.1 mg %) Range Mean % abnormality IgG Index (0.6-0.7) Range Mean % abnormality Oligoclonal IgG bands % abnormality NMR % abnormality

MS remission Other neurologic (n= 11) disorders (OND) (n=8)

3-8 4.8 25

19 3.8 18

1-7 3.1 25

1.3-10.6 4.6 75

1.5-7.3 3.7 81

0.9 7.9 3.4 62

0.6-1.8 1.0 75

0.4-1.4 0.7 27

0.44).8 0.5 25

75

27

0

100

82

0

ropathies, 1 leukodystrophy, t leukoencephalopathy, 1 cerebral ischemia) were used as controls. The N G F content of human CSF was measured using a 2-site ELISA, modified from that previously described [1]. In brief: 96-microtiter plates (Nunc) were coated with 50/A/well of 0.02/.tg/ml of monoclonal mouse a n t i - N G F 21/27 (Boehringer) diluted in 0.05 M carbonate buffer (pH 9.6). To assess the non-specific binding, parallel wells were coated with equal amounts of purified mouse I g G (Zymed). After an overnight incubation at room temperature the coating buffer was replaced with a blocking buffer (coating buffer + 1% BSA), 100/.d/well for at least 4 h at r o o m temperature. CSF samples were diluted 1:1 (v/v) with sample buffer 0.2% Triton X-100 (100 m M Tris-HCl p H 7.2,400 m M NaC1, 4 m M EDTA, 0.2 m M PMSF, 0.2 m M benzethonium chloride, 2 m M benzamidine, 40 U/ml aprotinin, 0.05% sodium azide, 2% BSA and 0.5% gelatin). After washing (50 m M TrisHC1 p H 7.2, 200 m M NaCI, 0.25% gelatin, 0.05% sodium azide and 0.1% Triton X-100), 50/d/well of standard and sample solution were distributed into the wells and left at room temperature (22°C) overnight. The N G F standard

solutions ranged between 1 100 pg of NGF. After extensive washings (at least 1 h) and incubation for 2 h at 37 ° C with 4 mU/well anti-fl-NGF galactosidase, 100/tl of substrate solution (4 mg of Chlorophenol red/ml substrate buffer: 100 m M HEPES, 150 m M NaC1, 2 m M MgC12, 0.1% sodium azide and 1% BSA) were added to each well and the plates incubated for 2 h at 37°C. The optical density was measured at 575 nm using an E L I S A reader (Dynatech) and the values of standards and samples were corrected by subtraction of the background value due to non-specific binding. Specificity for N G F was assessed using recombinant h u m a n N G F (Genentech), biologically active N G F from conditioned media of neuroblastoma cells (SH-SY5Y), and conditioned media from cultures of human skin fibroblasts stimulated with IL-lfl as standards. Recombinant brain-derived neurotrophic factor (BDNF) is not recognized in our assay at concentrations up to 20 ng/ml. Furthermore, biologically active B D N F protein in conditioned media from cultures of BDNF-transfected COS cells did not cross react with the antibody used in the ELISA. However, we cannot rule out that other neurotrophins, such as neurotrophin 3 (NT-3) and neurotrophin 4 (NT-4) [13], may be recognized by the antibodies used. We first measured the N G F content in human CSF in newborns, children and adults. More than 80 CSFs were divided into groups according to age and sex. While no significant difference between males and females was detected, N G F levels do seem to be closely related to age. As shown in Fig. 1, the CSF from newborns and children

30

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6.24 rots

2-6 yrs

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20-80

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Age Fig. 1. NGF content in normal human CSF from newborns, children and adults without inflammatory disease. Early in postnatal development the NGF level peaks and then decreases until it is almost undetectable in adults. The values for each group represent the mean of at least 15 CSF samples tested in triplicate _+standard error of the mean (S.E.M.).

ll 20-

O1 10 Ii

z

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__L.__L-Control

Acute attack

Remission

Fig. 2. NGF levels in the CSF of patients with MS. NGF content has been measured in patients in acute attacks (n=4), in remission (n=l 1) and in controls (n=8). All patients with acute attacks showed increased NGF levels compared with those of controls, while during remission the NGF content in CSF decreases. The values represent the mean of triplicate measures obtained in 3 independentexperiments.The difference between the mean values is significant (P<0.001) according to Student's t-test.

of up to 6 months (mean age of 2.5 months) shows the highest measured N G F levels, with a mean value of 23 pg/ml. During postnatal development the N G F content gradually decreases in CSF and is almost undetectable in adults. Because the N G F level in healthy adults seems to be extremely low, we wondered whether the N G F content in CSF would be altered by inflammation in the CNS. We have used CSF from 15 patients with MS. Since a characteristic feature of MS is recurrent attacks of inflammation we measured the N G F content in CSF in different clinical phases of the disease (acute attack and in remission). CSFs from 8 patients with other neurological diseases (OND) have been used as controls. N G F was detectable in all CSFs of MS patients analyzed (Fig. 2). Interestingly, N G F levels closely follow the phases of the disease. During acute attacks, patients exhibit a significant increase of N G F content (mean value 16.2 pg/ ml) when compared to controls. In contrast during remission the mean N G F levels in the CSF markedly decrease (mean value 5.6 pg/ml). These results strongly indicate that the N G F content follows the inflammatory response. This is supported by our recent data showing that patients with viral and bacterial meningitis express elevated N G F levels of CSF (LBL and UO, unpublished results). The strict age-dependency of N G F content in human CSF confirms its importance in the development of CNS neurons. During early mammalian development N G F expression is maximal in certain brain areas [15, 21]; it then decreases or becomes undetectable but could

be re-expressed at sites of lesion or after injury [10, 19]. Increased expression of N G F at sites of injury seems to be essential for neuronal recovery, and may also modulate the local immune response. Inflammatory stimuli enhance N G F expression, and N G F seems to act as an immunomodulatory cytokine stimulating lymphocyte proliferation and immunoglobulin production [25]. In the present study we have found increased N G F levels in the CSF of patients in acute phase MS, while during remission N G F in CSF markedly declines. Thus, there is a good correlation between the inflammatory episodes o f the disease and increased N G F production. N G F could be used for clinical characterisation of the inflammatory phase of MS. It is worth noting that in experimental allergic encephalomyelitis (EAE), an animal model for MS, both N G F m R N A and protein increase in the inflamed brain tissues [5]. Although the mechanisms controlling N G F expression are unclear, recent reports demonstrate that increased levels of cytokines such as IL-1, IL-6 and TNF-ct stimulate N G F expression. Increased levels o f these cytokines have been found in the CSF of MS patients [6, 7, 20, 28] and they could be responsible for the enhanced N G F production we have observed. The authors wish to thank Dr. M. Falcone (Department of Neurological Science, Universit& 'La Sapienza', Rome), Dr. R. Frei and Dr. J. Muser (Kantonsspital Basel) and Mrs. sc nat. G. Schlapfer (Kinderspital Basel) for providing CSF samples. The authors thank Dr. J. Scully for critical reading o f the manuscript. This study was supported by 'Associazione Italiana Sclerosi Multipla', by C N R 'Progetto Finalizzato Biotecnologie e Biostrumentazione: Sottoprogetto Biofarmaci' to Prof. R. Levi-Montalcini and Dr. L. Aloe, by Swiss National Foundation for Scientific Research (Grant 31-2995490), by Deutsche Forschungsgemeinschaft (SFB 325) and from B M F T (Grant 01 K L 8904/6) to Prof. U. Otten.

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