CSF findings in adrenoleukodystrophy: Correlation between measures of cytokines, IgG production, and disease severity

CSF Findings in Adrenoleukodystrophy: Correlation Between Measures of Cytokines, IgG Production, and Disease Severity John P. Phillips, MD*, Lawrence A. Lockman, MD*, Elsa G. Shapiro, PhD*, Bruce R. Blazar, MDt, Daniel J. Loes, MD*, Hugo W. Moser, MD #, and William Krivit, MD, PhD* The childhood-onset cerebral form of adrenoleukodystrophy has a devastating neurologic prognosis. Unfortunately, there is no early method of distinguishing it from the more benign forms of adrenoleukodystrophy, such as adrenomyeloneuropathy. To evaluate the manner in which this disease entity may be reflected in the cerebrospinal fluid, we studied a consecutive series of 19 patients, all with biochemically proved adrenoleukodystrophy. Total protein, immunoglobulin production, cytokine levels, and cerebrospinal fluid pressure were measured. In this single sample of cerebrospinai fluid, a significant correlation existed between clinical stage of the illness and cerebrospinal fluid myelin basic protein. No correlation existed with total protein, cytokines, or measures of immunoglobulin production. Phillips JP, Lockman LA, Shapiro EG, Blazar BR, Loes D J, Moser HW, Krivit W. CSF findings in adrenoleukodystrophy: Correlation between measures of cytokines, IgG production, and disease severity. Pediatr Neurol 1994; 10:289-294.

Introduction

COCALD is marked by a striking central nervous system (CNS) inflammatory reaction. Autopsy specimens disclosed areas of demyelination containing perivascular lymphocytic infiltration, consisting primarily of T-cells and macrophages [7]. Cytokines have also been demonstrated within these areas, consistent with a humoral immune response. Few studies, however, have evaluated the inflammatory component of cerebral spinal fluid (CSF) in patients with ALD. In 1975, Schaumburg et al. reported CSF findings in a series of 17 patients with clinical and pathological diagnosis of A L D [8]; they reported that CSF protein was elevated in almost all patients, while IgG was elevated in 5 of 10 patients. Another study of CSF in patients with ALD was conducted by Griffin et al. in 1985 [9]; they reported that 4 of 8 patients had an elevated IgG index and 5 of 6 had an elevated IgG. This report describes CSF data from a series of 19 patients with A L D at presentation for bone marrow transplant evaluation. In addition to the standard measures of protein, cells, and IgG production, this is the first series to report on the level of tumor necrosis factor alpha (TNF~x), interferon gamma (IFN~/), and interleukin-1 (IL-lot) in CSF of these patients prior to therapy.

Methods

Adrenoleukodystrophy (ALD) is an X-linked disease associated with elevated very long-chain fatty acids (VLCFA) [1,2]. A deficiency of lignoceroyl-CoA ligase activity results in disordered peroxisomal oxidation [3,4]. Of the 6 subtypes identified, all have similar biochemical profiles despite enormous clinical differences [5,6]. These subtypes vary from the childhood-onset cerebral form (COCALD), which results in a rapidly progressive dementia, to individuals who remain asymptomatic throughout life. Unfortunately, no method exists to differentiate between the different subtypes of A L D until clinical symptoms, magnetic resonance imaging (MRI) changes, or neuropsychologic changes occur.

The study populationconsisted of 19 patients with ALD referred to the University of Minnesota for evaluation for possible bone marrow transplantation. All had elevated serum levels of VLCFA and were 5-15 years of age. Patients includedprobands as well as affected, but asymptomatic, brothers. Medication at initial evaluation generally included physiologic doses of steroids and a diet of restricted VLCFA with supplemental "Lorenzo's oil" (i.e., glyceryl trierucate and glyceryl trioleate). CSF was obtained as part of this initial evaluation. In addition to the usual studies (i.e., protein, glucose, cell count, culture), spinal fluid was evaluated for myelin basic protein (MBP), oligoclonalbands, IgG, IgG synthesis, IgG index, TNFot, IFN3,, and IL-lct. All laboratory tests were conducted at the University of Minnesota's clinical laboratory. Total protein and glucose were assayed using the Kodak Ektachem 700-XR. Serum and CSF IgG were obtained with the

From the *Division of Pediatric Neurology; Departments of tPediatrics and ~Radiology;Universit~ of Minnesota Medical School; Minneapolis, Minnesota;"Kennedy Krieger Institute; Baltimore, Maryland.

Communications should be addressed to: Dr. Phillips; Box 486; 420 Delaware Street Southeast; Minneapolis, MN 55455-0323. Received December 29, 1993; accepted March 7, 1994.

© 1994 by Elsevier Science Inc. • 0887-8994/94/$7.00

Phillips et al: Cytokines in Adrenoleukodystrophy 289

Table 1. Patient Number

Clinical presentation Age (yr, mo)

Neuropsychometrics

MRI Posterior disease Loes score = 9 Posterior disease Loes score = 10

Medications

I Q F S = 108, V = 101, P = 115 Poor auditory processing I Q F S = 114, V = 115, P = 109 Normal verbal and nonverbal reasoning, attention and emotion; auditory perception difficulties I Q F S = 76, V = 72, P = 85 Compulsive, deteriorating language function

Cortisone 0.5 mg/kg/day z 4 years; diet x 5 years Cortisone 0.6 mg/kg/day x 27 months; diet x 27 months

Normal

Diet x 18 months

I Q F S = 90, V = 93, P = 89 Poor fine motor coordination; difficulties in visual processing, auditory processing, visual motor integration, and arithmetic I Q F S = 103, V = 107, P = 100 Visual processing deficit I Q F S = 91, V = 90, P = 95 Visual perceptual difficulties

Hydrocortisone O. 75 mg/kg/day x 1 year; diet x 1 year

Hyperactive deep tendon reflexes, L > R Clumsy fine motor coordination, L > R Moderate gross and fine motor delay

ALD1M

10, 9

ALD2M

9, 8

ALD3M

11, 7

Posterior disease Loes score = 11

ALD5M

5, 3

Posterior disease Loes score = 0.5

ALD6M

9, 10

ALD7M

5, 3

Posterior disease Loes score = 0.5 Normal Loes score = 0

ALD8M

9, 10

Posterior disease Loes score = 4

I Q F S = 109, V = 114, P = 102 Visual and auditory processing difficulties, new learning problems

ALDIOM

15, 0

ALD 11M

9, 3

Frontal disease t o e s score = 5 Posterior disease Loes score = 14

I Q F S = 81, V = 75, P = 91 Frontal lobe syndrome IQ V = 76 Severe visual and auditory agnosia

ALD12M

9, 1

Posterior disease Loes score = 9

I Q F S = 84, V = 91, P = 80 Severe visual processing deficit, deaf

None

ALD14M

7, 7

Frontal disease Loes score = 15.5

Diet x 1 month

ALD15M

5, 1

ALDI6M

8, 8

Posterior disease Loes score = 4 Normal Loes score = 0

I Q F S = 69, V = 76, P = 65 Severe attention-deficit disorder, memory and visual processing deficits I Q F S = 103, V = 107, P = 98 Poor visual processing I Q F S = I I 6 , V = I I 2 , P = 117 Hyperactive

ALDI7M

9, 10

Frontal disease Loes score = 8

ALD18M

12, 2

Frontal disease Loes score = 12

ALDI9M

7, 8

Posterior disease Loes score = 22

ALD22M

7, 0

Frontal disease Loes score = 5

ALD23M

15, 1

Posterior disease Loes score = 11

IQ v = 87 (Peabody Picture Vocabulary test in Spanish), P = 62 Attention-deficit disorder; poor motor coordination, memory, and visual integration skills I Q F S = 75, V = 85, P = 68 Hyperactive, poor new learning and frontal lobe function I Q F S = 70, V = 87, P = 58 Markedly impaired motor skills; increasing visual perceptual difficulties IQ V = 90 Attention-deficit disorder; severe motor impairment

I Q F S = 84, V = 81, P = 88 Visual processing difficulties (continued)

290

PEDIATRIC N E U R O L O G Y

Examination

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Normal

Diet x 3 years

Normal examination

Cortisone 0.5 mg/kg/day x 5 months; diet only since birth including Lorenzo's oil x 5 months Hydrocortisone 0.5 mg/kg/day x 3 years, fiudrocortisone 0.1 mg/day (0.0025 mg/kg/day) x 3 years; diet x 3 months Diet x many years, poor compliance Diet x 3 months

Mild hypotonia

Mild hypotonia, pale optic discs

Mild hypotonia Poor fine motor and gross motor coordination Hearing impaired, LE hyporeflexia, optic pallor, occasional incontinence Extensor plantar reflexes, poor gross motor skills, pale optic discs

Diet z 2 months

Normal

Hydrocortisone 0.4 mg/kg/day x 6 months; diet × 6 months Prednisone 0.16 mg/kg/day x 10 months; diet × 10 months

Normal (twin brother in vegetative state at patient presentation) Hyperactive

Cortisone 0.5 mg/kg/day x 1 month

Normal

Methylphenidate 1 mg/kg/day

Choreoathetosis, dysarthria, dysmetria, lead-pipe rigidity

Methylphenidate 0.25 mg/kg/day, imipramine 0.5 mg/kg/day

Dysarthria, poor coordination, dysmetria, generalized hypotonia with toe-walking Complex partial seizures (under good control); normal examination

Cortisone 0.3 mg/kg/day, carbamazepine 20 mg/kg/day

Table 1.

(continued)

Patient Number

(yr, ran)

ALD25M

10, 9

Age

Abbreviations: FS = Full scale

MRI

Frontal disease Loes score = 6

Neuropsychometrics

Medications

I Q F S = 52, V = 59, P = 53 Attention-deficit disorder, rapid downhill course

LE = Lower extremity

Beckman Array analyzer. IgG synthesis, believed to be an accurate reflection of brain IgG production, was derived using the formula by Tourtellotte [10]: [5] [(IgGcsF - IgGs/369) - (AlbcsF - Albs/230)(IgGs/Albs)(0.43)] IgG index is a measure of CSF IgG that is derived from brain (rather than coming from serum through a disrupted blood-brain barrier) and is obtained as follows [11]: IgG index = (CSF IgG/CSF albumin) (Serum albumin/Serum IgG) MBP was performed by ARUP Labs using a radioimmunoassay kit (DSL1500) from Diagnostic System Laboratories. The lowest level of detection (95% confidence level) with this kit is 0.26 ng/ml. Oligoclonal bands were also assayed by ARUP Labs utilizing an Iso-lab Isoelectric Focusing Kit (Resolve CSF FR8065). We were able to assay IFN% IL-let, and TNF~t as specimen quality and quantity would permit. To assay TNFct accurately, we found it essential to freeze samples immediately after obtaining them and to thaw them just once prior to the assay. Quantitation of TNF~t was evaluated using Boehringer Manneheim's TNFct ELISA kit (Indianapolis), IFN~/ was measured in Endogen's IFN~/ELISA kit (Boston), and I L - l a was measured using R & D System's IL-lct Quantikine ELISA kit (Minneapolis). The level of sensitivity for each assay is as follows: TNFct, < 10 pg/ml; IFN% < 1 0 pg/ml; and IL-la, < 5 . 0 pg/ml. The normal level is considered to be 0.0 for all cytokine assays. Each patient also was evaluated by a pediatric neuropsychologist and a pediatric neurologist, and had cranial MRI performed.

Results Table 1 summarizes the clinical data. All 19 patients had MRI scans performed on initial evaluation; 11 revealed posteriorly predominant white matter changes, 2 were normal, and 6 had disease primarily in the frontal lobes. No scans had evidence of cortical or brainstem involvement. The Loes Scale is a method of quantifying MRI changes in patients with ALD [12]. The scale is based on the location and extent of disease, ranging from 0 (normal MRI) to 34 (extensive diffuse neurologic disease). Neuropsychometric data were obtained on all patients as well. CSF results were not known at testing. Full Scale IQ scores ranged from 116 to 5 2 : 6 had scores > 100, 7 were 80-100, and 6 were < 80. Patients with frontal disease on MRI had associated behavioral changes typical of frontal lobe dysfunction on neuropsychometric testing. Abnormal CSF data are summarized in Table 2. CSF leukocyte count was 3/1~1 or less in all patients. There

None

P = Performance

Examination Poor fine motor and gross motor coordination, self-stimulating behavior

V = Verbal

was no significant correlation between disease severity (as measured by Full Scale IQ) and age, oligoclonal bands, total protein, IgG, IgG synthesis, IgG index, CSF pressure, IFN% or IL-lct. As an additional measure of disease severity, all patients were staged clinically according to the method described by Shapiro and Klein [13] which utilizes a scale of 0 to 4 based on such clinical criteria as school performance, behavior difficulties as reported by parents, and neurologic examination abnormalities. As expected, there was a strong correlation between the clinical stage and IQ. Six patients had accurate measurements of TNFct, 3 of which were abnormally elevated. Although these numbers are too small to draw any definite conclusions, no clear correlation existed between TNFa and Full Scale IQ in our patients. Of the various proteins measured in spinal fluid, 9 patients (48%) had at least one abnormal value. As can be seen in Table 2, the 2 most severely involved children (ALDI4M and ALD25M) had the greatest number of abnormal values. When 2 patients unable to have full neuropsychologic testing because of physical handicaps were excluded from the data, a significant correlation was found between MBP and IQ. An inverse relationship exists between MBP and Full Scale IQ (P = .017, correlation coefficient = - 0 . 5 8 ; Fig 1A), MBP and Verbal IQ (P = . 109, correlation coefficient = - 0 . 4 ; Fig 1B), and MBP and Performance IQ (P = .001, correlation coefficient = - 0 . 7 ; Fig IC). No significant correlation exists between MBP and total protein, measures of IgG production, MRI Loes score, or age of the patient.

Discussion The clinical course of patients cannot be predicted on the basis of VLCFA levels; it ranges from COCALD with rapid progression and fatal dementia to adrenomyeloneuropathy (AMN) which may remain asymptomatic for years. Neither is family history predictive, as both COCALD and the more benign AMN form can exist within the same kindred [5,6,14-16]. The inability to diagnose COCALD before CNS symptoms occur makes timely therapeutic intervention difficult. As part of the pathologic picture of COCALD, there is

Phillips et al: Cytokines in Adrenoleukodystrophy

291

Table 2.

Abnormal CSF data

CSF

Patient Number

Age (yr, mo)

ALD3M ALD7M ALDI2M ALD14M ALDI7M ALD19M ALD22M ALD23M ALD25M Normal

11, 5, 9, 7, 9, 7, 7, 15, 10,

Clinical Stage

7 3 1 7 l0 8 0 l 9

IQ

2 l 2 4 3 3 3 3 4 0

Abbreviations: FS = Full Scale

FS FS FS FS FS FS v FS FS FS

IQ IQ IQ IQ IQ IQ IQ IQ IQ IQ

= = = = = = = = = =

2

a._ 1.5

0.5

0

A

50

60

70

50

60

70

50

60

70

80 90 Full Scale IQ

100

110

120

100

110

120

100

110

120

2,5

Z

,S

1.5 1

c_ =E

0.5

0

B

80 Verbal

90 IQ

2.5

Z

m ~

I[ 0.5

0

C

80 90 P e r f o r m a n c e IQ

Figure 1. (A) Myelin basic protein versus Full Scale IQ. (B) Myelin basic protein versus Verbal IQ. (C) Myelin basic protein versus Performance IQ.

292

Protein

Myelin Basic Protein

<20 <20 < 20 44.5 33 24 < 20 <20 24 < 20 cm H20

34 < 10 59 155 22 < 10 32 22 94 15-60 mg/dl

0.3 <0.2 0.7 1.8 2.4 1.0 3.6 0.8 2.0 < 4 . 0 ng/ml

V = Verbal

2.5

c

76 91 84 69 87 70 90 84 52 100

Opening Pressure

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a marked inflammatory response in the CNS [ 17]. Plaques of demyelination have been found in the brains of patients with COCALD which contain numerous macrophages and T-cells [7,8]. These cells are involved in propagating and modulating the cellular immune response through such secretory products as cytokines which have been associated with myelin damage in vitro [18]. Of the cytokines we were able to measure, 6 of 6 patients had normal IL- 1et, 11 of 12 had normal IFN~, and 3 of 6 had normal TNFct levels. These data suggest that CSF IL-h~ and IFN'y do not reflect disease progression. TNFoc also does not appear to correlate with disease severity, although more patients are required to answer this question. We also measured CSF protein and IgG production. Two of 19 patients had elevated CSF protein, 3 of 13 patients had abnormally increased IgG, IgG index, and IgG synthesis, and 1 of 13 patients had only an elevated IgG index. None of these CSF parameters reflected disease severity. These findings are in contrast with previous reports [8,9] and suggest that modulators of immune function other than IgG may be important in COCALD. All patients had CSF pressure measured, 6 of 19 of which were abnormally elevated. Although the CSF pressure did not correlate with disease severity, it is unclear why any patients had elevated pressure. It is known that in certain storage diseases, such as mucopolysaccharidoses, a product of abnormal metabolism interferes with reabsorption of CSF through the arachnoid granulation tissue, causing hydrocephalus. This is not known to occur in ALD. Further study is required to demonstrate what role, if any, increased CSF pressure plays in the pathophysiology of ALD. MBP is a component of normal myelin sheath and is elevated in other demyelinating diseases, such as multiple sclerosis or postmeasles encephalitis [ 19]. All our patients had normal MBP (using adult standards; no normal values are available for pediatric patients). Within this normal range, however, there was significant correlation between disease severity (as measured by Full Scale IQ, Perfor-

CSF Oligoclonal

Bands Positive Negative Negative Negative Negative Negative Negative Negative Positive Negative

IgG

IgG Synthesis

IgG Index

TNFce

IFN-/

Interleukin-lce

3.7 0.6 11.4 23.1 3.3 1.0 1.9 2.4 6.7 <4.5 mg/dl

0.0 0.0 21.0 49.0 4.2 0.0 0.4 0.0 9.7 <8.0 mg/dl

0.46 0.50 0.75 0.77 0.79 0.42 0.66 0.55 0.65 0.28-0.66

----0.0 2.7 12.3 2.9 0.0 0.0 pg/ml

0.0 4.5 0.0 0.0 0.0 0.0 0.0 -0.0 0.0 pg/ml

----0.0 0.0 0.0 0.0 0.0 0.0 pg/ml

mance IQ, or Verbal IQ) and MBP. Although it is unclear what initiates the demyelinating inflammatory response in COCALD, our data indicate that this demyelination is reflected in elevations of CSF MBP, and may be a useful marker of disease progression. In addition, it may help differentiate the early stages of the COCALD from more benign forms of the disease. Of all the parameters studied, most were abnormal in 2 patients whose clinical, neuropsychologic, and neuroradiologic courses were severe. One patient (ALD 14M) was markedly affected on presentation to our clinic and has since died. The other patient (ALD12M) has done poorly and continues to manifest disease progression. A third patient (ALD22M) had clinically advanced disease and the highest TNFet measured although all other CSF values were normal; he has done poorly despite recent bone marrow transplantation. Of the other 16 patients, clinical presentation and CSF findings varied considerably. Because of the obvious problems with brain biopsy in living patients, a more reasonable marker of disease progression may lie within the CSF. Our series of 19 patients is the largest to date that examines the CSF findings in patients with biochemically proved ALD. We are continuing our systematic follow-up of these patients. Serum VLCFA levels do not correlate with disease progression; therefore, we are currently assessing the relationship between CSF VLCFA levels and clinical status. Other relationships being explored include analyzing NMR spectroscopy data on these patients and the use of pentoxyphylline to inhibit TNFot. Although previous work has clearly demonstrated a marked inflammatory reaction in the CNS of patients with active ALD, for unclear reasons this was not consistently reflected in the CSF of our patients. Those with far advanced disease tended to have some CSF abnormalities, but there was no specific pattern identified, and few abnormalities were observed in the CSF of less involved patients. The finding, not previously reported, of a significant relationship between MBP and disease severity is not

surprising given the known demyelination that occurs in this disease. Ongoing studies are systematically examining the CSF changes over time. As patients are subjected to various treatment modalities, such as pentoxiphylline (which interferes with the action of TNFet), we hope to identify an early marker of COCALD, as well as biochemical parameters that may accurately reflect disease progression. We thank Mary Rich-Halet of the University of Minnesota Cytokine Reference Laboratory for performing cytokine assays. This study was supported in part by NINDS NS29099. BRB is a scholar of the Edward Mallinckrodt, Jr. Foundation.

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