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Aids-associated vacuolar myelopathy and tumor necrosis factor-alpha (TNFα)
JOURNAL OF THE
NEUROLOGICAL SCIENCES Journal of the Neurological
Science5
I38 (1996)
I35-
144
Aids-associated vacuolar myelopathy and tumor necrosis factor-alpha CTNF~ S.V. Tan ‘, R.J. Guiloff a.*, D.C. Henderson ‘, B.G. Gazzard ‘, R. Miller d
Received
29 June 1995: revissd
14 November
1995: accepted 24 November
1995
Abstract The spinal cords from 15 patients with AIDS-associated vacuolar myelopathy (VM), 4 AIDS patients without VM, and 5 HIV-seronegative controls. were studied with immunocytochemistry for TNFcu. CSF and blood from HIV-seropositive patients with VM (II = 161, non-vacuolar myelopathies (n = 8). CNS infection but no clinical myelopathy t/z = 3 I I, no clinical or radiological evidence of CNS disease (n = 9), and from 7 HIV-seronegative controls with motor neurone disease were assayed for TNFa using an ELISA technique. TNFa was present on immunostaining in all the I5 cords with VM studied. The stained cells were macrophages, microglia and endothelial cells. The amount of immunostainiq was higher in cords with VM compared with cords from HIV-seropositive patients without VM ( p = 0.001 I. The distribution of staining corresponded to the areas of pathology but did not correlate with the severity of the VM. Immunostaining was also higher in the HIV-seropositive group compared to the HIV-seronegative controls ( p = 0.001). There was no significant difference in the levels of TNFa in the CSF of patients with VM compared to any of the other groups studied. Blood levels of TNFa were lower in the HIV-seropositive controls without CNS disease and in the HIV-seronegative MND controls, than in patients with VM. non-vacuolar myelopathies, and CNS disease. CSF TNFa levels did not appear to be a reliable indicator of intramedullary levels. The findings support the hypothesis that TNFQ may be relevant in the pathogenesis of vacuolar change in VM. Krn~~~rr/.v: HIV I: AIDS:
Myelopathy:
Spinal cord disease: Paraparesis:
TNF:
1. Introduction Vacuolar myelopathy is a common cause of disability in patients with AIDS. being reported in up to 557~ (Artigas et al.. 1990) of AIDS post mortems, with most series reporting an incidence of 22-33s (Petit0 et al., 1985; Navia et al.. 1986; Petit0 et al., 1986; Grafe and Wiley, 1989). Clinically it is characterized by a subacute or chronic progressive spastic paraparesis, sometimes associated with upper motor neurone bladder involvement and a sensory ataxia. Histologically there are intramyelinic and periaxonal vacuoles (Maier et al., 1989) containing, or closely associated with. macrophages within the lateral, posterior and, less commonly, anterior columns of the spinal cord (Petit0 et al.. 1985). The pathology is largely
Corresponding author. Present address: Regional Neurosciencea Centre, Charing Cross Hospital. Fulham Palace Road. London W6 8RF. UK. Tel: f54 181.716 8319: fax: +34 181-746 8420. 0022-510X/96/$15.00 0 I996 Elsevier SD/ 0022-S 10X(95)00354I
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Cytokines:
Cerehrospinal
fluid
concentrated in the thoracic and cervical cord, with relative sparing of the lumbar segments. The histological appearances resemble those of subacute combined degeneration of the cord seen in vitamin B I2 deficiency (Petit0 et al., 1985). The pathogenetic mechanisms involved in VM remain unknown. Most pathological series have not found an association between the presence of low serum vitamin B12 levels and VM (Petit0 et al., 1985; Rhodes et al., 1989; Petit0 et al., 1994) although a higher prevalence of B I2 deficiency or malabsorption in AIDS patients with myelopathy has been reported (Kieburtz et al., 1991). In addition, abnormalities in methyl group metabolism, such as are seen in vitamin B12 and folate deficiency, have also been found in patients with AIDS (Surtees et al., 1990b; Keating et al., 1991). The possibility that abnormalities in B12 related metabolic pathways may contribute to the myelin damage in VM cannot be excluded. There is no conclusive evidence of a direct causal
relationship between the presence of HIV in the spinal cord and the development of VM. Some authors have found an association between the presence of detectable virus and VM (Maier et al.. 1989; Rhodes et al., 1989; Eilbott et al., 1989; Budka, 1990; Weiser et al., 19901, whilst others have not (Artigas et al., 1990; Grafe and Wiley, 1989; Rosenblum et al., 1989; Kure et al.. 1991: Scaravilli et al., 1992; H&in et al., 1992; Bergmann et al., 1993; Petit0 et al., 1994). Of 30 reported cases of VM in which HIV was detected (Grafe and Wiley, 1989; Maier et al., 1989; Eilbott et al., 1989; Rosenblum et al., 1989; Sharer et al.. 1990; Budka. 1990; Weiser et al., 1990; Scaravilli et al., 1992; H&in et al., 1992; Petit0 et al.. 1994), multinucleated giant cells were described in 25, suggesting that positive staining for HIV may relate to concomitant HIV myelitis. The possibility of an indirect role of HIV in the pathogenesis of VM, however, remains. A central role for macrophages in the pathogenesis of VM is suggested by their presence within, and closely associated to the vacuoles (Petit0 et al.. 1985). Several macrophage derived products have been shown to be neurotoxic (Heyes et al., 199 I; Giulian et al.. 1990), but one in particular, TNFa, has been noted for its myelinotoxic properties (Selmaj and Raine, 1988) and has been implicated in the pathogenesis of demyelination in multiple sclerosis (Hofman et al., 1989, Selmaj et al., 1991, Cannella and Raine, 1995). It is possible that in VM, TNFa produced within the spinal cord by macrophages may mediate the vacuolar change. Our demonstration. in a previous morphometric study (Tan et al., I99.5), of an early prominent presence of activated microglia and macrophages in VM lends some support to this view.
We have looked for immunocytochemical evidence of the presence of TNFcx within the spinal cord, and investigated levels of TNFa in the CSF and blood, using an ELISA technique in patients with vacuolar myelopathy and in HIV-seropositive and -seronegative controls.
2. Materials
and methods
The spinal cords were obtained from in-patients who died between 1991 and 1994. 2. I. 1. Patierlts There were I.5 patients with ~~~ud~~r m~elopcrthy. aged 25-53 years (median 40 years). All were male homosexuals with AIDS. All had been seen clinically by the authors; nine had clinical signs of myelopathy. Concomitant spinal cord and brain infections are given in Table I. There were 4 HIV-seropositi1.e umtro1.r \cithout WI. All were male homosexuals with AIDS. Further details are given in Table 2. There were 5 HIV-.serot1eSNtil.e c.orltro1.s without myeloppathy. The causes of death were head injury (21, bronchopneumonia (I ), metastatic adenocarcinoma ( I ) and undetermined (I) (Table 3). 2.1.2. Tissue samples and reagents Blocks of thoracic spinal cord obtained from patients at post mortem were snap frozen to -70°C. There was no significant difference in the mean post mortem delay be-
Table I TNFa immunostaining in spinal cords of patients with vacuolar myelopat .h) Pt
Age Sex
PM delay (h)
I
27M 35 M 41M 40 M 29 M 53M 52 M 44M 47 M 25 M 40 M 49 M 32 M 28M 44 M
12 72 96 71 24 72 96 24 71 I20 71 72 96 24 48
2
3 4 5 6 7 8 9 IO II I2 I3 I4 I5
TNFa
Location
Cells staining with TNFol
sev
+++
sev sev sev sev he”
++ ++ ++ ++ ++ ++ ++ if ++
LC. PC. AC LC PC, LC PC. LC. AC PC > LC PC, LC. AC LC. PC. AC LC. PC LC, PC LC, AC LC. AC. PC Diffuse WM > GM LC, PC, AC LC PC(medial)LC
mat. micgl. endoth mat. endoth. mat. micgl mat. micgl. endoth mat, micgl. endoth mat. micgl. endoth mat. micgl mat, micgl mat. miql. endoth mat. micgl. endoth mat, micgl micgl. endoth mat. micfl mat. micgl. endoth mat, micgl
VM
mod mod mod mod mod mld mld mld mld
++ ++ ++
Spinal Cord HIVpY
Other Spinal Cord pathology
-
+ -
HIV myel
-
MGN myel
HIV myel HIV myel
Brain patholoSy HIVE. asp HIVE. HiVlep CMVE PBL NSG CMVE HIVlep. FPL CMVE Emboli HIVE HIVE HIVE HIVE. PML HIVE HIVE
Pt = patient; M = male: PM = post mortem; sev = severe: mod = moderate; mld = mild: LC = lateral columns: PC = posterior column>: AC = anterior columns: mat = macrophage: micgl = micro_rlia: endoth = endothelial cell; - = negative: + = positive: HIVE = HIV encephalitis: ahp = aspergillosi~: HIV myel = HIV myelitis: HIVlep = HIV leukoencephalopathy: CMVE = cytomegalovirus encephalitis: PBL = primary brain lymphoma: PML = progressive multifocal leukoencephalopathy; NSG = non-specific mild gliosis: FPL = focal pontine leukoencephalopathy; MGN myel = microglial nodule myelitis: WM = white matter: GM = grey matter.
S. V. Tan et al. /Journal
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Table 2 TNFo immunostaining in spinal cords of HIV seropositive patients without VM Pt
Age Sex
PM delay (hours)
Spinal cord pathology
TNFcv
Location
Cells positive with TNFo
SC HIV p24
Brain pathology
16 17 18 19
46M 33M 57M 44M
72 72 24 72
microglial microglial microglial microglial
+ (inflam) + (micgl) ++-
Diffuse Discrete foci Discrete foci Discrete cells
micgl, endoth micgl micgl, endoth endoth
-
NSG NSG PML normal
activation and inflam activation activation activation
-
lnflam = inflammation: SC = spinal cord; Other abbreviations as in Table I
tween the patients with VM (mean 68 h, SD 28.6) and the nine controls (mean 54.7 h, SD 28.8)(Table 1, 2 and 3). Additional spinal cord blocks and brain blocks were fixed in 10% buffered formalin for paraffin embedding. Five pm paraffin sections were processed using routine stains including haematoxylin and eosin (H and E), Luxol Fast Blue/Cresyl Violet (LFB/N) for myelin and neurons respectively, and Glees/Marsland method for axons. Staining with the lectin Ricinus communis agglutinin (RCA, VECTOR Laboratories, UK, dilution 1:3000) was used for microglia/monocyte-macrophages. Immunocytochemical staining, using the modified ABC method (Sobel et al., 19841, was performed for HIV p24 (DAKO, UK, monoclonal antibody, dilution 1:80), glial fibrillary acid protein (GFAP. DAKO, UK, polyclonal, dilution 1:400) for glial fibrils, L26 (DAKO, UK, monoclonal, dilution 1500) for B cells, and UCHL- 1 (DAKO, UK, monoclonal, dilution I:lOOO> for T cells. Ten Frn frozen sections of spinal cord were cut onto poly-L-lysine-coated slides. Sections were air-dried for 30 min, fixed in cold acetone at -20°C for 20 min, and then air dried overnight. As a positive control for the staining of TNFa, U-937 monocytes were stimulated to produce TNF~Y with phorbol myristate acetate (PMA) for 4 h, pelleted onto a slide, and immunostained as outlined below. Sections treated identically but omitting the primary antibody served as negative controls. Immunostaining was performed using a polyclonal rabbit anti-TNFo (Sigma), developed using recombinant human TNFcr as immunogen, at a dilution of 1:250.
secondary antibody (biotinylated swine anti-rabbit (DAKO)) for 45 min. Following incubation with ExtrAvidin-alkaline phosphatase conjugate (SIGMA) for 30 min, colour was developed using a Vector blue alkaline phosphatase substrate kit III (VECTOR), and conterstained with nuclear fast red.
2. I .3. TNFcv immunostaining Slides were incubated with normal swine serum (DAKO) for 20 min, followed by the primary antibody for 60 min at room temperature. They were incubated with the
2.2. CSF and blood TNFcv levels
2.1.4. Classification of secerity of uacuolar myelopathy Pathological changes were evaluated on the paraffin embedded tissue. At least 3 levels of spinal cord (high, mid/low thoracic and lumbar) were studied in each patient. The cervical cord was available in 5 patients. Vacuolar myelopathy was qualitatively classified as absent, mild, moderate and severe according to accepted criteria (Rosenblum et al., 1989) (Fig. 1). Classification was based on the worst affected areas of the cord. Six cords were classified as severe VM, 5 as moderate and 4 as mild. The morphometric findings in the cords of patients I, 2, 3, 7, 8 and 9 are published elsewhere (Tan et al., 1995). 2.1.5. Analysis of amount of TNFa immunostaining The amount of positive staining with TNFcv was assessed by a blinded assessor, and graded as negative (-Xno staining), very mild (+ - )(few cells, most faintly stained), mild ( + )(moderate numbers of cells, most faintly stained), moderate (+ + Xmoderate amount of discrete cells, some strongly stained), and severe ( + + + )(large numbers of cells with some confluent areas strongly stained) (Figs. 2 and 3, Tables 1 and 2, and 3).
This clinical series was collected from in-patients who underwent CSF examination as part of their routine inves-
Table 3 TNFcv Immunostaining in spinal cords of HIV seronegative controls Pt
Age Sex
PM delay (hours)
Spinal cord pathology
TNFCr
20 21 22 23 24
71 73 37 48 39
72 12 96 24 48
none microglial activation none none none
+ -
F M M M F
F = female; Other abbreviations as per Table I
Location
Cells positive with TNFcv
scattered
micgl
Brain pathology ischaemic changes intracranial haematoma extradural haematoma subdural haematoma normal
S.V. Tan et ul. /Journal
of the Neurological Sciences 138 (I 996) 134-144
tigations between August 1991 and August 1993; their blood was also taken for serum or plasma estimation of TNFcY.
137
2.2.1. Patients Sixteen had a clinical diagnosis of cacuolar myelopathy, defined as a subacute or chronic paraparesis or tetra-
Fig. I. Photomicrographs of thoracic cord sections with (a) moderate (patient lo), and (b) severe VM (patient lKLFB/cresyl
violet
X
12).
paresis in an HIV-seropositive patient, in whom there was no clinical, radiological or laboratory evidence of other pathology sufficient to account for the signs. Fifteen had MRIs of the spinal cord. All had extensor plantar responses and lower limb hyperreflexia. Sixteen had pyramidal weakness; 2 had spasticity but no weakness. Thirteen had upper motor neurone bladder symptoms, 8 had a gait ataxia, and I I patients had posterior column sensory loss.
Their median age was 39.5 years (range 34 - 50). All were male homosexuals. Fifteen had AIDS, I had ARC. Vacuolar myelopathy was confirmed pathologically in 5/5 post mortems from this group. 2.2.2. Controls There were 48 HIV-seropositire controls. Eight of them had non-cacuolar myeloputhies. Their median age was
Fig. 2. Grades of TNFcI immunostaining (blue) in the cytoplasm (see Methods). Nuclei are counterstained with nuclear fast red. Most cell5 shown are macrophages or microglia. (a) very mild (+ - ) (patient 18): (b) mild ( + ) (patient 17); (c) moderate ( + + ) (patient 7): Cd) severe ( + + + ) (patient I ): and (e) negative control. ( X 93).
Fig.3
Fig.4
Fig. 3. Grades of TNFa immunostaining (as in Fig. 2) shown at lower power. (a) mild (+ Xpatient 17) (X 68); (b) moderate ( + + Xpatient 7); (c) severe ( + + + Xpatient I )( X 27). Fig. 4. Morphology of cell types staining for TNFLu. (a) “foamy” vessels. arrow). ( X 109).
macrophage (arrow); (b) microglial cell (arrow); (c) endothelial cells (lining blood
140
XV. Turnet al. /Journal
qf the Neurological Sciences 138 (1996) 134-144
35.5 years (range 24-43). All were male homosexuals with AIDS. Clinically, 4 had CMV myeloradiculitis (1 patient confirmed pathologically), and 1 CMV myelitis. One had herpes zoster myeloradiculitis. One had neurosyphilis, and another had a transient encephalomyelopathy of uncertain aetiology. Thirty one had CNS infection but no clinical myelopathy. Their median age was 37 years (range 26-57). Thirty had AIDS, 1 had ARC. Twenty eight were male homosexuals, 2 were female intravenous drug users, and 1 was a female without known risk factors. Fifteen had encephalitis, 7 had cryptococcal meningitis, 4 had progressive multifocal leukoencephalopathy (PML), 2 had cerebral toxoplasmosis, 2 had viral meningitis and 1 had cerebral aspergillosis. The diagnosis was confirmed at post mortem in this last patient, and in 1 of the cases with PML. Nine controls had no clinical or radiological ecidence of CNS disease. Their median age was 34 years (range 31-55). Eight had AIDS, 1 had ARC. All were male homosexuals. There were 7 HIV-seronegatice controls with sporadic motor new-one disease (MND) or amyotrophic lateral sclerosis (ALS). Their median age was 51 years (range 39-66). All were male. All had clinical evidence of spinal cord involvement: all had upper and lower motor neurone signs in the lower limbs as well as lower motor neurone signs in the upper limbs. Six also had upper motor neurone signs in the upper limbs. Six had extensor plantar responses. Two also had bulbar signs. 2.2.3. CSF and blood samples The 4th ml of CSF was collected in sterile containers, and frozen at - 70°C. Blood samples were collected simultaneously in endotoxin free sterile tubes, separated and stored at -70°C. All samples and standards were pretreated with /3-propiolactone prior to assay. Blood and CSF samples were assayed for TNFa by a standard ELISA technique (British Biotechnology, human TNFa Quantikine assay). The minimum detectable level was 4.4 pg/ml in calibrator diluent for CSF and blood. The maximum TNFa level in serum and plasma samples collected from apparently healthy normal donors (n = 40) assayed by the manufacturers was 15.6 pg/ml. 2.3. Statistics The Mann Whitney-U test was used to compare TNFa immunostaining between cords with and without myelopathy. Spearman’s rank coefficient ( rs) was used for correlations between severity of VM and grade of immunostaining for TNFcu. The Kruskall-Wallis one-way analysis of variance was used to compare CSF and blood TNFcv levels in the groups studied. The unpaired t-test was used to compare post mortem delay between patients with and without VM.
3. Results 3.1. Immunocytochemistry for TNFcx in the spinal cord See Tables 1-3, and Figs. 2-4. The main finding was the presence of abundant TNFcv immunostaining in macrophages within the areas of vacuolar change in the cords with VM. The cells staining positive were morphologically macrophages and microglia, although staining was also seen in some endothelial cells. The amount of immunostaining for TNFcr, did not correlate with the pathologic severity of VM (r, = 0.41, p = 0.13); reflecting the observation that TNFcv was present in conspicuous amounts even in mild-moderate VM. Staining for TN& was however, significantly higher in cords with VM compared with HIV-seropositive AIDS controls and HIVseronegative cords without VM (Mann-Whitney U, p = 0.00004). This observation still held (p = O.OOl), if the HIV-seronegative group was excluded, since this group would be expected to have low levels of TNFcx. TNFcv staining was significantly higher in the HIV-seropositive group as a whole compared with the HIV-seronegative cords (p = 0.001). In the cords with VM, the distribution of TNFa staining corresponded with areas of pathology; most of the positive-staining macrophages were situated adjacent to and within vacuoles in the lateral, posterior and anterior columns. In the cases of mild VM, staining was prominent in activated microglia and macrophages in the lateral and posterior columns, despite the presence of relatively few vacuoles. In the HIV-seropositive AIDS control cords, one showed diffuse staining in activated microglia, and 3 showed discrete positivity in occasional microglial cells. The location of the cells in these cords did not correspond to the areas normally involved by VM. In the HIVseronegative controls, staining was only seen in a few microglial cells in one cord, and was markedly less than that seen in the HIV-seropositive cords. In patient 3, spinal cord tissue as well as CSF and blood were available; there was no elevation in blood or CSF TNFa levels despite the strong immunopositivity demonstrated in the cord. 3.2. HIV immunostaining HIV p24 immunostaining was positive in 3/15 spinal cords of patients with VM, and in none of the 4 HIVseropositive AIDS control cords. In all three cords staining positive for HIV, multinucleated giant cells were also present, suggesting the presence of concomitant HIVmyelitis. 3.3. CSF and blood TNFo lecels 3.3.1. Patients. Vacuolar myelopathy (n = 16) TNFa was detectable in the CSF of only one patient with VM. In the blood, the TNFcx was detectable in 12
S.V. Tan et ul./Journal
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patients; median 10.7 pg/ml (range O-292.9 pg/ml). In the 5 patients with post mortem confirmation, TNFa was undetectable in CSF in all, and was detected in the blood in three (37.2, 11.4, 9.3 pg/ml) (Table 4). 3.3.2. HIV-seropositil’e
controls
3.3.2.1. Non-cacuolur myelopathies (n = 8). CSF TNFcY was undetectable in 5 patients. In the remaining 3, the CSF TNFcv levels were 9, 11 and 46 pg/ml. In the blood, TNFu was detectable in 6 patients; median 19.25 pg/ml (range O-673.6 pg/ml). 3.3.2.2. CNS infection without clinical myelopathy (n = 31). CSF TNFcx was undetectable in 21 patients. In the remaining 10, the median CSF TNFL~ was 49.5 pg/ml (range 24.2 to 72.4 pg/ml). In the blood, TNFcv was
141
detectable in 14 patients; median 45.2 pg/ml 352.2 pg/ml).
(range O-
3.3.2.3. No clinical or radiological ecidence of CNS disease fn = 9). CSF TNFa was undetectable in 7 patients. In the remaining 2, the levels were 20.2 and 61.4 pg/ml. In the blood, TNFar was detectable in only 1 patient (9.3 pg/ml). 3.3.3. HIV-seronegatiue controls with motor neurone disease fn = 7) CSF TNFcv was undetectable in 4 patients. In the remaining 3, the levels were 61.4, 64.1 and 77 pg/ml. TNFcv was undetectable in all the blood samples in this group. The results were analysed using the Kruskal-Wallis one-way analysis of variance as the data were not normally
Table 4 CSF and blood levels of TNFa in patients with vacuolar myelopathy and HIV seropositive and HIV seronegative controls Vacuolar myelopathy
HIV seropositive controls Non-vacuolar myelopathies
Pt
Id 2 3 4” 5 6 7” 8 9 10 II I2 I3 14” 15” I6
CSF TNFcx
Blood TNFcx
(pg/mO
(pg/mO
0 0 0 0 69.6 0 0 0 0 0 0 0 0 0 0 0
0 292.9 51.8 31.2 0 0 0 7.5 12.8 II 10.5 12.7 9.7 II.4 9.3 10.9
Pt
CSF TNFcv
Blood TNFa
(pg/ml)
(pg/ml)
I7 18” I9 20 21 22 23 24
46. I 0 0 0 Il.6 0 0 9
673.6 31.2 0 0 10.4 IO 9.1 9.7
HIV seronegative controls CNS infection. No myelopathy
No CNS disease
Pt b
Pt
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 d 49 50 51 52 53 54 55’
CSF TNF~Y
Blood TNFLV
h/ml)
(pg/ml)
0 0 44.4 0 72.4 0 0 0 0 0 0 0 0 0 0 50.3 54.9 24.2 48.6 46.3 0 0 48.2 53.6 0 0 0 54.5 0 0 0
0 62.5 225.1 0 45.1 0 0 0 15 0 1.5 10.1 IO.9 IO 0 87.8 352.2 45.7 96.7 0 0 0 44.8 0 0 0 0 89 0 0 0
56 57 58 59 60 61 62 63 64
Motor neurone disease
CSF TNFcv
Blood TNFa
(pg/ml)
(pg/ml)
0 0 0 20.2 61.4 0 0 0 0
0 0 0 0 0 0 0 0 9.3
Pt TNFCV 65 66 67 68 69 70 71
CSF
61.4 0 II 64.1 0 ,o 0
Blood
0 0 0 0 0 0 0
Pt = patient; CSF = cerebrospinal fluid; TNFcv = tumor necrosis factor alpha; a Post mortem confirmation of diagnosis; h Clinical diagnosis as follows: Patients 25-39 = encephalitis, 40-46 = cryptococcal meningitis, 47-50 = progressive multifocal leukoencephalopathy, 5 l-52 = cerebra] toxoplasmosis, 53-54 = viral meningitis; 55 = cerebral aspergillosis.
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S.V. Tan et al. / Journal of the Neurological Sciences 138 (19961 134-144
distributed. There was no difference in the CSF TNFa levels (p = 0.25) but blood TNFa levels were significantly different ( p = 0.005) between the groups. The latter were higher in the patients with VM, non-vacuolar myelopathy, and CNS infection without myelopathy compared with the HIV-seropositive patients without CNS disease and HIV-seronegative patients with motor neurone disease. 3.4. Serum 812 and folate
In the pathological series, B12 levels were low in l/4 patients with VM tested. Red cell and serum folate levels were normal. In the clinical series, B12 levels were low in 3/16 patients with VM, and serum folate levels low in l/ 16 patients. Red cell folate levels were normal.
4. Discussion Our results show that TNFo is present in significant amounts in macrophages, microglia and endothelial cells within the areas of pathology in VM. In contrast to Tyor et al (Tyor et al., 1993) who recently reported the presence of TNFa! in spinal cords with VM by immunocytochemistry, we found increased amounts of TNFa in cords with VM compared to cords from HIV-seropositive patients without VM. The amount of TNFo staining did not correlate with the severity of the VM. This may be due to the fact that in mild VM the activated microglia contained conspicuous amounts of TNFcu. These microglia may be the precursors of some of the TNFcr staining macrophages seen in the lesions of more advanced VM (Tan et al., 1995). The presence of abundant TNFcv in mild, presumably early lesions would support the view that it may play a role in the myelin vacuolation seen in VM. Our finding of an increased amount of TNFcv staining in spinal cords from HIV-seropositive patients compared to HIV-seronegative normal controls concurs with the results of Tyor et al. (Tyor et al., 1993). The staining in these cords was mainly in microglia and endothelial cells, and may suggest a background of immune activation within the CNS of HIV-seropositive patients. It is also possible that some of these cords may represent the very early stages of VM. The presence of TNFcv within the spinal cord of patients with VM was not reflected by increased levels of TNFcv in the CSF. Similarly increased TNFcr in brain tissue has been found in multiple sclerosis (Selmaj et al., 1991) though in CSF it has been reported as both absent (Gallo et al., 1989) and elevated (Sharief and Hentges, 1991). TNFo has a short half-life in biological fluids (Selby et al., 1987). CSF TNFa levels could therefore be expected to be mildly, or only modestly, elevated in conditions where it is produced largely within the
parenchyma of the CNS itself rather than in the meninges. In our patients, as in a previous report (Mastroianni et al., 1992) TNFa was most frequently detected in the CSF of patients with cryptococcal meningitis (5/7). The higher blood levels of TNFa in the patients with VM, with non-vacuolar myelopathy, and those with CNS disease, compared with HIV-seropositive patients without CNS disease and with HIV-seronegative patients with motor neurone disease may suggest a concomitant systemic immune response in the former conditions. The proportion of patients with AIDS in the HIV-seropositive without CNS disease group was similar to the other groups. Thus, although a general immune activation might be expected in patients with advanced AIDS it would not, by itself, account for the elevated blood TNFa levels found in the former groups. The group of patients with MND were older than the other groups; however, TNFo levels have not been shown to decrease significantly with age (Riancho et al., 1994). Our results suggest that locally produced TNFa may play a role in the myelin damage and vacuolation seen in VM. Its presence in the lateral and posterior columns in mild, presumably early, VM and its capacity to produce ballooning of myelin in cultures of mouse spinal cord tissue (Selmaj and Raine, 1988) suggest that TNFcv may be directly involved in the pathogenesis of the vacuolar change. It is not known why myelin vacuolation occurs in such a distinctive distribution within the thoracic cord. The processes associated with chronic macrophage activation and myelin damage within the spinal cord may have some bearing on the anatomic distribution of the pathological change in VM. One hypothesis is that in the late stages of HIV infection, immune activation within the CNS, either due to the presence of HIV encephalitis or opportunistic infections, may lead to the local production of myelin/membrane damaging cytokines such as TNFa (Tyor et al., 1993) toxins, or oxygen radicals by microglia, resident macrophages and endothelial cells. Damaged or degenerating myelin fibres may act to attract scavenging macrophages. Those macrophages expressing gp120 may further damage myelin by binding to sulphatide and myelin associated glycoprotein on oligodendrocyte membranes (van den Berg et al., 1992). The local presence of TNFa may result in an upregulation of MHC Class II antigen expression on microglia and macrophages (Sherry and Cerami, 1988). These cells, having ingested and processed myelin protein, may then act as antigen presenting cells thus further enhancing the immune reaction. Such chronic myelin damage is likely to lead to increased demands for metabolic products required for repair. Critical to these repair mechanisms is the methylation of fatty acids, phospholipids, polysaccharides and proteins, such as myelin basic protein, by the body’s universal methyl donor (S-adenosylmethionine), a product of the methyl transfer pathway involving B 12 and folate metabolism (Surtees and Hyland, 1990a). As this is the
S.V. Tan et al. /Journal
of the Neurological Sciences 138 i 19961 134- 144
sole pathway for the generation of methyl groups, should the consumption of methyl groups for repair mechanisms become excessive, a secondary methyl group deficiency similar to that seen in vitamin B 12 and folic acid de% ciency may develop (Surtees et al., 1990b), thus predisposing the spinal cord to myelin degeneration similar to that seen in subacute combined degeneration of the cord (SACD)(Hyland et al., 1988). Such a cellular deficiency may account for the close histological resemblance of VM to SACD without a demonstrable association with reduced serum B12 and folate levels, a finding we have also confirmed. Further myelin degeneration may attract more macrophages and perpetuate the process.
Acknowledgements This work was supported by the North West Thames Regional Health Authority and the Special Trustees of the Westminster and Roehampton Hospitals. We thank Professor F Scaravilli from the Institute of Neurology for his advice and for providing facilities for the immunocytochemistry. We are also grateful to Dr A. Ciardi and Mr A. Beckett for technical help, and to Dr J.N. Harcourt-Webster and Dr S. Lucas for performing the post mortem examinations.
References Artigas, J.. Grosse, Cl. and Niedobitek. F. (1990) Vacuolar myelopathy in AIDS. A morphological analysis, Pathol. Res. Practice 186: 228-237. Bergmann, M.. Gullotta, F.. Kuchelmeister, K., Masini, T. and Angeli, G. (1993) AIDS-myelopathy. A neuropathological study. Pathol. Res. Practice 189: 58-65. Budka, H. (1990) Human immunodeficiency virus (HIV) envelope and core proteins in CNS tissues of patients with the acquired immune deficiency syndrome (AIDS). Acta Neuropathol. 79: 61 I-619. Cannella, B. and Raine, C.S. (199.5) The adhesion molecule and cytokine profile of multiple sclerosis lesions. Ann. Neural. 37: 424-435. Eilbott. D.J., Peress, N.. Burger, H., LaNeve, D., Orenstein, J., Gendelman. H.E., Seidman, R. and Weiser, B. (1989) Human immunodeficiency virus type I in spinal cords of acquired immunodeficiency syndrome patients with myelopathy: expression and replication in macrophages. Proc Nat1 Acad Sci USA 86: 3337-3341. Gallo, P., Piccinno. M.G., Krzalic, L. and Tavolato, B. (1989) Tumor necrosis factor alpha (TNF alpha) and neurological diseases. Failure in detecting TNF alpha in the cerebrospinal fluid from patients with multiple sclerosis. AIDS dementia complex. and brain turnours. J. Neuroimmunol. 23: 41-44. Giulian, D., Vaca. K. and Noonan, C.A. (1990) Secretion of Neurotoxins by Mononuclear Phagocytes infected with HIV-l. Science 250: 1593-1596. Grafe, M.J. and Wiley. CA. (1989) Spinal cord and peripheral nerve pathology in AIDS: the roles of cytomegalovirus and human immunodeficiency virus. Ann Neural 25: 561-566. H&tin, D., Smith, T.W., De Girolami, U., Sughayer, M. and Hauw, J.J. (1992) Neuropathology of the spinal cord in the acquired immunodeficiency syndrome. Hum Pathol 23: I 106-I I 14. Heyes, M.P., Brew, B.J., Martin, A., Price, R.W.. Salazar, A.M., Sidtis, J.J.. Yergey, J.A., Mouradian, M.M., Sadler, A.E., Keilp, J. and et al.,
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(1991) Quinolinic acid in cerebrospinal fluid and serum in HIV-I infection: relationship to clinical and neurological status. Ann Neural 29: 202-209. Hofman, F.M., Hinton, D.R., Johnson, K. and Merrill. J.E. (1989) Tumor necrosis factor identified in multiple sclerosis brain. J. Exp. Med. 170: 607-612. Hyland, K.. Smith, I.. Bottiglieri, T., Perry. J.. Wendel. U., Clayton, P.T. and Leonard, J.V. (1988) Demyelination and decreased S-adenosylmethionine in 5, IO- methylenetetrahydrofolate reductase deficiency. Neurology 38: 459-462. Keating, J.N., Trimble, K.C., Mulcahy, F., Scott, J.M. and Weir, D.G. ( 199 I) Evidence of brain methyltransferase inhibition and early brain involvement in HIV-positive patients. Lancet 337: 935-939. Kieburtr, K.D., Giang, D.W., Schiffer, R.B. and Vakil, N. (1991) Abnormal vitamin B I2 metabolism in human immunodeficiency virus infection. Association with neurological dysfunction. Arch. Neurol. 48: 312-314. Kure, K., Llena, J.F., Lyman, W.D., Soeiro, R., Weidenheim, K.M., Hirano, A. and Dickson, D.W. (1991) Human immunodeficiency virus-1 infection of the nervous system: an autopsy study of 268 adult, pediatric, and fetal brains. Hum. Pathol. 22: 700-710. Maier, H., Budka, H., Lassmann, H. and Pohl, P. (1989) Vacuolar myelopathy with multinucleated giant cells in the acquired immune deficiency syndrome (AIDS). Light and electron microscopic distribution of human immunodeficiency virus (HIV) antigens. Acta Neuropathol. 78: 497-503. Mastroianni, C.M., Paoletti, F.. Valenti, C., Vullo, V., Jirillo, E. and Delia, S. (1992) Tumour necrosis factor (TNF-alpha) and neurological disorders in HIV infection. J. Neural. Neurosurg. Psychiatry 55: 219-221. Navia, B.A., Cho. E-S.. Petito, C.K. and Price, R.W. (1986) The AIDS Dementia Complex: II. Neuropathology. Ann. Neurol. 19: 525-535. Petito, C.K., Cho. E-S,, Lemann, W.. Navia, B.A. and Price, R.W. (1986) Neuropathology of acquired immunodeficiency syndrome (AIDS): an autopsy review. J. Neuropathol. Exp. Neurol. 45: 635-646. Petito. C.K., Navia, B.A., Cho, E.S., Jordan. B.D., George, D.C. and Price. R.W. (1985) Vacuolar myelopathy pathologically resembling subacute combined degeneration in patients with the acquired immunodeficiency syndrome. N. Eng. J. Med. 312: 874-879. Petito, C.K.. Vecchio, D. and Chen, Y-T. (1994) HIV antigen and DNA in AIDS spinal cords correlate with macrophage infiltration but not with vacuolar myelopathy. J. Neuropathol. Exp. Neural. 53: 86-94. Rhodes, R.H., Ward, J.M., Cowan, R.P. and Moore. P.T. (1989) Immunohistochemical localization of human immunodeficiency viral antigens in formalin-fixed spinal cords with AIDS myelopathy. Clin. Neuropathol. 8: 22-27. Riancho, J.A.. Zarrabeitia, M.T.. Amado, J.A.. Olmoa, J.M. and Gonzalez-Macias, (1994) Age related differences in cytokine secretion, Gerontology 40: 8- 12. Rosenblum, M.. Scheck, A.C., Cronin. K., Brew, B.J.. Khan, A., Paul, M. and Price, R.W. (1989) Dissociation of AIDS-related vacuolar myelopathy and productive HIV-1 infection of the spinal cord. Neurology 39: 892-896. Scaravilli, F., Sinclair, E., Arango, J-C., Manji, H.. Lucas, S. and Harrison. M.J.G. (1992) The pathology of the posterior root ganglia in AIDS and its relationship to the pallor of the gracile tract. Acta Neuropathol. 84: l63- 170. Selby, P., Hobbs, S.. Viner, C., Jackson, E., Jones. A., Newell, D., Calvert, AH., McElwain, T., Fearon, K., Humphreys, J. and Shiga, T. (1987) Tumour necrosis factor in man: clinical and biological observations. Br. J. Cancer 56: 803-808. Selmaj, K., Raine, C.S., Cannella, B. and Brosnan, C.F. (1991) Identification of lymphotoxin and tumor necrosis factor in multiple sclerosis lesions. J. Clin. Invest. 87: 949-954. Selmaj, K.W. and Raine, C.S. (1988) Tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro. Ann. Neural. 23: 339346.
Sharer. L.R.. Dowling. P.C., Michaels, J., Cook, S.D., Menonna, J., Blumberg, B.M. and Epstein. L.G. (1990) Spinal cord disease in children with HIV-I infection: a combined molecular biological and neuropathological study. Neuropathol. Appl. Neurobiol. 16: 317-33 I. Sharief, M.K. and Hen&e\. R. (I 99 I) Association between Tumor Necrosis Factor-a and disease progression in patients with multiple sclerosis. N. Eng. J. Med. 325: 467-472. Sherry. B. and Cerami. A. (1988) Cachectin/tumor necrosis factor exerts endocrine, paracrine. and autocrine control of inflammatory responses. J. Cell Biol. 107: 1269-1277. Sobel. R.A.. Blanchette, B.W.. Bhan. A.K. and Calvin, R.B. (1984) The immunopathology of experimental allergic encephalomyelitis. Quantitative analysis of inflammatory cells in situ. J. Immunol. 132: 23932401. Surteea, R. and Hyland. K. (1990a) Cerebrospinal fluid concentrations of S-Adenosylmethionine. methionine, and 5-methyltetrahydrofolate in a reference population: cerebrospinal fluid S-adenosylmethionine declines with age in humans. Biochem. Med. Metab. Biol. 44: 192-199.
Surtees. R.. Hyland, K. and Smith, I. (1990b) Central-nervous-system methyl-group metabolism in children with neurological complications of HIV infection. Lancet 335: 619-621. Tan, S.V., Guiloff, R.J. and Scaravilli, F. (1995) AIDS-associated vacuolar myelopathy - a morphometric study. Brain I 18: 1247-1261. Tyor. W.R., Glass, J.D., Baumrind. N., McArthur, J.C., Griffin, J.W.. Becker, P.S. and Griffin. D.E. (1993) Cytokine expression of macrophages in HIV- I -associated vacuolar myelopathy. Neurology 43: 1002-1009. van den Berg, L.H.. Sadiq, S.A., Lederman. S. and Latov. N. (1992) The gpl20 Glycoprotein of HIV-l binds to sulfatide and to the myelin associated glycoprotein. J. Neurosci. Res. 33: 5 13-5 18. Weiser, B.. Peres N., La Neve. D.. Eilbott. D.J., Seidman. R. and Burger, H. (1990) Human immunodeficiency virus type I expression in the central nervous system correlates directly with extent of disease. Proc. Natl. Acad. Sci. USA 87: 3997-4001.
NEUROLOGICAL SCIENCES Journal of the Neurological
Science5
I38 (1996)
I35-
144
Aids-associated vacuolar myelopathy and tumor necrosis factor-alpha CTNF~ S.V. Tan ‘, R.J. Guiloff a.*, D.C. Henderson ‘, B.G. Gazzard ‘, R. Miller d
Received
29 June 1995: revissd
14 November
1995: accepted 24 November
1995
Abstract The spinal cords from 15 patients with AIDS-associated vacuolar myelopathy (VM), 4 AIDS patients without VM, and 5 HIV-seronegative controls. were studied with immunocytochemistry for TNFcu. CSF and blood from HIV-seropositive patients with VM (II = 161, non-vacuolar myelopathies (n = 8). CNS infection but no clinical myelopathy t/z = 3 I I, no clinical or radiological evidence of CNS disease (n = 9), and from 7 HIV-seronegative controls with motor neurone disease were assayed for TNFa using an ELISA technique. TNFa was present on immunostaining in all the I5 cords with VM studied. The stained cells were macrophages, microglia and endothelial cells. The amount of immunostainiq was higher in cords with VM compared with cords from HIV-seropositive patients without VM ( p = 0.001 I. The distribution of staining corresponded to the areas of pathology but did not correlate with the severity of the VM. Immunostaining was also higher in the HIV-seropositive group compared to the HIV-seronegative controls ( p = 0.001). There was no significant difference in the levels of TNFa in the CSF of patients with VM compared to any of the other groups studied. Blood levels of TNFa were lower in the HIV-seropositive controls without CNS disease and in the HIV-seronegative MND controls, than in patients with VM. non-vacuolar myelopathies, and CNS disease. CSF TNFa levels did not appear to be a reliable indicator of intramedullary levels. The findings support the hypothesis that TNFQ may be relevant in the pathogenesis of vacuolar change in VM. Krn~~~rr/.v: HIV I: AIDS:
Myelopathy:
Spinal cord disease: Paraparesis:
TNF:
1. Introduction Vacuolar myelopathy is a common cause of disability in patients with AIDS. being reported in up to 557~ (Artigas et al.. 1990) of AIDS post mortems, with most series reporting an incidence of 22-33s (Petit0 et al., 1985; Navia et al.. 1986; Petit0 et al., 1986; Grafe and Wiley, 1989). Clinically it is characterized by a subacute or chronic progressive spastic paraparesis, sometimes associated with upper motor neurone bladder involvement and a sensory ataxia. Histologically there are intramyelinic and periaxonal vacuoles (Maier et al., 1989) containing, or closely associated with. macrophages within the lateral, posterior and, less commonly, anterior columns of the spinal cord (Petit0 et al.. 1985). The pathology is largely
Corresponding author. Present address: Regional Neurosciencea Centre, Charing Cross Hospital. Fulham Palace Road. London W6 8RF. UK. Tel: f54 181.716 8319: fax: +34 181-746 8420. 0022-510X/96/$15.00 0 I996 Elsevier SD/ 0022-S 10X(95)00354I
Science B.V.
All rights reserved
Cytokines:
Cerehrospinal
fluid
concentrated in the thoracic and cervical cord, with relative sparing of the lumbar segments. The histological appearances resemble those of subacute combined degeneration of the cord seen in vitamin B I2 deficiency (Petit0 et al., 1985). The pathogenetic mechanisms involved in VM remain unknown. Most pathological series have not found an association between the presence of low serum vitamin B12 levels and VM (Petit0 et al., 1985; Rhodes et al., 1989; Petit0 et al., 1994) although a higher prevalence of B I2 deficiency or malabsorption in AIDS patients with myelopathy has been reported (Kieburtz et al., 1991). In addition, abnormalities in methyl group metabolism, such as are seen in vitamin B12 and folate deficiency, have also been found in patients with AIDS (Surtees et al., 1990b; Keating et al., 1991). The possibility that abnormalities in B12 related metabolic pathways may contribute to the myelin damage in VM cannot be excluded. There is no conclusive evidence of a direct causal
relationship between the presence of HIV in the spinal cord and the development of VM. Some authors have found an association between the presence of detectable virus and VM (Maier et al.. 1989; Rhodes et al., 1989; Eilbott et al., 1989; Budka, 1990; Weiser et al., 19901, whilst others have not (Artigas et al., 1990; Grafe and Wiley, 1989; Rosenblum et al., 1989; Kure et al.. 1991: Scaravilli et al., 1992; H&in et al., 1992; Bergmann et al., 1993; Petit0 et al., 1994). Of 30 reported cases of VM in which HIV was detected (Grafe and Wiley, 1989; Maier et al., 1989; Eilbott et al., 1989; Rosenblum et al., 1989; Sharer et al.. 1990; Budka. 1990; Weiser et al., 1990; Scaravilli et al., 1992; H&in et al., 1992; Petit0 et al.. 1994), multinucleated giant cells were described in 25, suggesting that positive staining for HIV may relate to concomitant HIV myelitis. The possibility of an indirect role of HIV in the pathogenesis of VM, however, remains. A central role for macrophages in the pathogenesis of VM is suggested by their presence within, and closely associated to the vacuoles (Petit0 et al.. 1985). Several macrophage derived products have been shown to be neurotoxic (Heyes et al., 199 I; Giulian et al.. 1990), but one in particular, TNFa, has been noted for its myelinotoxic properties (Selmaj and Raine, 1988) and has been implicated in the pathogenesis of demyelination in multiple sclerosis (Hofman et al., 1989, Selmaj et al., 1991, Cannella and Raine, 1995). It is possible that in VM, TNFa produced within the spinal cord by macrophages may mediate the vacuolar change. Our demonstration. in a previous morphometric study (Tan et al., I99.5), of an early prominent presence of activated microglia and macrophages in VM lends some support to this view.
We have looked for immunocytochemical evidence of the presence of TNFcx within the spinal cord, and investigated levels of TNFa in the CSF and blood, using an ELISA technique in patients with vacuolar myelopathy and in HIV-seropositive and -seronegative controls.
2. Materials
and methods
The spinal cords were obtained from in-patients who died between 1991 and 1994. 2. I. 1. Patierlts There were I.5 patients with ~~~ud~~r m~elopcrthy. aged 25-53 years (median 40 years). All were male homosexuals with AIDS. All had been seen clinically by the authors; nine had clinical signs of myelopathy. Concomitant spinal cord and brain infections are given in Table I. There were 4 HIV-seropositi1.e umtro1.r \cithout WI. All were male homosexuals with AIDS. Further details are given in Table 2. There were 5 HIV-.serot1eSNtil.e c.orltro1.s without myeloppathy. The causes of death were head injury (21, bronchopneumonia (I ), metastatic adenocarcinoma ( I ) and undetermined (I) (Table 3). 2.1.2. Tissue samples and reagents Blocks of thoracic spinal cord obtained from patients at post mortem were snap frozen to -70°C. There was no significant difference in the mean post mortem delay be-
Table I TNFa immunostaining in spinal cords of patients with vacuolar myelopat .h) Pt
Age Sex
PM delay (h)
I
27M 35 M 41M 40 M 29 M 53M 52 M 44M 47 M 25 M 40 M 49 M 32 M 28M 44 M
12 72 96 71 24 72 96 24 71 I20 71 72 96 24 48
2
3 4 5 6 7 8 9 IO II I2 I3 I4 I5
TNFa
Location
Cells staining with TNFol
sev
+++
sev sev sev sev he”
++ ++ ++ ++ ++ ++ ++ if ++
LC. PC. AC LC PC, LC PC. LC. AC PC > LC PC, LC. AC LC. PC. AC LC. PC LC, PC LC, AC LC. AC. PC Diffuse WM > GM LC, PC, AC LC PC(medial)LC
mat. micgl. endoth mat. endoth. mat. micgl mat. micgl. endoth mat, micgl. endoth mat. micgl. endoth mat. micgl mat, micgl mat. miql. endoth mat. micgl. endoth mat, micgl micgl. endoth mat. micfl mat. micgl. endoth mat, micgl
VM
mod mod mod mod mod mld mld mld mld
++ ++ ++
Spinal Cord HIVpY
Other Spinal Cord pathology
-
+ -
HIV myel
-
MGN myel
HIV myel HIV myel
Brain patholoSy HIVE. asp HIVE. HiVlep CMVE PBL NSG CMVE HIVlep. FPL CMVE Emboli HIVE HIVE HIVE HIVE. PML HIVE HIVE
Pt = patient; M = male: PM = post mortem; sev = severe: mod = moderate; mld = mild: LC = lateral columns: PC = posterior column>: AC = anterior columns: mat = macrophage: micgl = micro_rlia: endoth = endothelial cell; - = negative: + = positive: HIVE = HIV encephalitis: ahp = aspergillosi~: HIV myel = HIV myelitis: HIVlep = HIV leukoencephalopathy: CMVE = cytomegalovirus encephalitis: PBL = primary brain lymphoma: PML = progressive multifocal leukoencephalopathy; NSG = non-specific mild gliosis: FPL = focal pontine leukoencephalopathy; MGN myel = microglial nodule myelitis: WM = white matter: GM = grey matter.
S. V. Tan et al. /Journal
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of the Neurological
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Table 2 TNFo immunostaining in spinal cords of HIV seropositive patients without VM Pt
Age Sex
PM delay (hours)
Spinal cord pathology
TNFcv
Location
Cells positive with TNFo
SC HIV p24
Brain pathology
16 17 18 19
46M 33M 57M 44M
72 72 24 72
microglial microglial microglial microglial
+ (inflam) + (micgl) ++-
Diffuse Discrete foci Discrete foci Discrete cells
micgl, endoth micgl micgl, endoth endoth
-
NSG NSG PML normal
activation and inflam activation activation activation
-
lnflam = inflammation: SC = spinal cord; Other abbreviations as in Table I
tween the patients with VM (mean 68 h, SD 28.6) and the nine controls (mean 54.7 h, SD 28.8)(Table 1, 2 and 3). Additional spinal cord blocks and brain blocks were fixed in 10% buffered formalin for paraffin embedding. Five pm paraffin sections were processed using routine stains including haematoxylin and eosin (H and E), Luxol Fast Blue/Cresyl Violet (LFB/N) for myelin and neurons respectively, and Glees/Marsland method for axons. Staining with the lectin Ricinus communis agglutinin (RCA, VECTOR Laboratories, UK, dilution 1:3000) was used for microglia/monocyte-macrophages. Immunocytochemical staining, using the modified ABC method (Sobel et al., 19841, was performed for HIV p24 (DAKO, UK, monoclonal antibody, dilution 1:80), glial fibrillary acid protein (GFAP. DAKO, UK, polyclonal, dilution 1:400) for glial fibrils, L26 (DAKO, UK, monoclonal, dilution 1500) for B cells, and UCHL- 1 (DAKO, UK, monoclonal, dilution I:lOOO> for T cells. Ten Frn frozen sections of spinal cord were cut onto poly-L-lysine-coated slides. Sections were air-dried for 30 min, fixed in cold acetone at -20°C for 20 min, and then air dried overnight. As a positive control for the staining of TNFa, U-937 monocytes were stimulated to produce TNF~Y with phorbol myristate acetate (PMA) for 4 h, pelleted onto a slide, and immunostained as outlined below. Sections treated identically but omitting the primary antibody served as negative controls. Immunostaining was performed using a polyclonal rabbit anti-TNFo (Sigma), developed using recombinant human TNFcr as immunogen, at a dilution of 1:250.
secondary antibody (biotinylated swine anti-rabbit (DAKO)) for 45 min. Following incubation with ExtrAvidin-alkaline phosphatase conjugate (SIGMA) for 30 min, colour was developed using a Vector blue alkaline phosphatase substrate kit III (VECTOR), and conterstained with nuclear fast red.
2. I .3. TNFcv immunostaining Slides were incubated with normal swine serum (DAKO) for 20 min, followed by the primary antibody for 60 min at room temperature. They were incubated with the
2.2. CSF and blood TNFcv levels
2.1.4. Classification of secerity of uacuolar myelopathy Pathological changes were evaluated on the paraffin embedded tissue. At least 3 levels of spinal cord (high, mid/low thoracic and lumbar) were studied in each patient. The cervical cord was available in 5 patients. Vacuolar myelopathy was qualitatively classified as absent, mild, moderate and severe according to accepted criteria (Rosenblum et al., 1989) (Fig. 1). Classification was based on the worst affected areas of the cord. Six cords were classified as severe VM, 5 as moderate and 4 as mild. The morphometric findings in the cords of patients I, 2, 3, 7, 8 and 9 are published elsewhere (Tan et al., 1995). 2.1.5. Analysis of amount of TNFa immunostaining The amount of positive staining with TNFcv was assessed by a blinded assessor, and graded as negative (-Xno staining), very mild (+ - )(few cells, most faintly stained), mild ( + )(moderate numbers of cells, most faintly stained), moderate (+ + Xmoderate amount of discrete cells, some strongly stained), and severe ( + + + )(large numbers of cells with some confluent areas strongly stained) (Figs. 2 and 3, Tables 1 and 2, and 3).
This clinical series was collected from in-patients who underwent CSF examination as part of their routine inves-
Table 3 TNFcv Immunostaining in spinal cords of HIV seronegative controls Pt
Age Sex
PM delay (hours)
Spinal cord pathology
TNFCr
20 21 22 23 24
71 73 37 48 39
72 12 96 24 48
none microglial activation none none none
+ -
F M M M F
F = female; Other abbreviations as per Table I
Location
Cells positive with TNFcv
scattered
micgl
Brain pathology ischaemic changes intracranial haematoma extradural haematoma subdural haematoma normal
S.V. Tan et ul. /Journal
of the Neurological Sciences 138 (I 996) 134-144
tigations between August 1991 and August 1993; their blood was also taken for serum or plasma estimation of TNFcY.
137
2.2.1. Patients Sixteen had a clinical diagnosis of cacuolar myelopathy, defined as a subacute or chronic paraparesis or tetra-
Fig. I. Photomicrographs of thoracic cord sections with (a) moderate (patient lo), and (b) severe VM (patient lKLFB/cresyl
violet
X
12).
paresis in an HIV-seropositive patient, in whom there was no clinical, radiological or laboratory evidence of other pathology sufficient to account for the signs. Fifteen had MRIs of the spinal cord. All had extensor plantar responses and lower limb hyperreflexia. Sixteen had pyramidal weakness; 2 had spasticity but no weakness. Thirteen had upper motor neurone bladder symptoms, 8 had a gait ataxia, and I I patients had posterior column sensory loss.
Their median age was 39.5 years (range 34 - 50). All were male homosexuals. Fifteen had AIDS, I had ARC. Vacuolar myelopathy was confirmed pathologically in 5/5 post mortems from this group. 2.2.2. Controls There were 48 HIV-seropositire controls. Eight of them had non-cacuolar myeloputhies. Their median age was
Fig. 2. Grades of TNFcI immunostaining (blue) in the cytoplasm (see Methods). Nuclei are counterstained with nuclear fast red. Most cell5 shown are macrophages or microglia. (a) very mild (+ - ) (patient 18): (b) mild ( + ) (patient 17); (c) moderate ( + + ) (patient 7): Cd) severe ( + + + ) (patient I ): and (e) negative control. ( X 93).
Fig.3
Fig.4
Fig. 3. Grades of TNFa immunostaining (as in Fig. 2) shown at lower power. (a) mild (+ Xpatient 17) (X 68); (b) moderate ( + + Xpatient 7); (c) severe ( + + + Xpatient I )( X 27). Fig. 4. Morphology of cell types staining for TNFLu. (a) “foamy” vessels. arrow). ( X 109).
macrophage (arrow); (b) microglial cell (arrow); (c) endothelial cells (lining blood
140
XV. Turnet al. /Journal
qf the Neurological Sciences 138 (1996) 134-144
35.5 years (range 24-43). All were male homosexuals with AIDS. Clinically, 4 had CMV myeloradiculitis (1 patient confirmed pathologically), and 1 CMV myelitis. One had herpes zoster myeloradiculitis. One had neurosyphilis, and another had a transient encephalomyelopathy of uncertain aetiology. Thirty one had CNS infection but no clinical myelopathy. Their median age was 37 years (range 26-57). Thirty had AIDS, 1 had ARC. Twenty eight were male homosexuals, 2 were female intravenous drug users, and 1 was a female without known risk factors. Fifteen had encephalitis, 7 had cryptococcal meningitis, 4 had progressive multifocal leukoencephalopathy (PML), 2 had cerebral toxoplasmosis, 2 had viral meningitis and 1 had cerebral aspergillosis. The diagnosis was confirmed at post mortem in this last patient, and in 1 of the cases with PML. Nine controls had no clinical or radiological ecidence of CNS disease. Their median age was 34 years (range 31-55). Eight had AIDS, 1 had ARC. All were male homosexuals. There were 7 HIV-seronegatice controls with sporadic motor new-one disease (MND) or amyotrophic lateral sclerosis (ALS). Their median age was 51 years (range 39-66). All were male. All had clinical evidence of spinal cord involvement: all had upper and lower motor neurone signs in the lower limbs as well as lower motor neurone signs in the upper limbs. Six also had upper motor neurone signs in the upper limbs. Six had extensor plantar responses. Two also had bulbar signs. 2.2.3. CSF and blood samples The 4th ml of CSF was collected in sterile containers, and frozen at - 70°C. Blood samples were collected simultaneously in endotoxin free sterile tubes, separated and stored at -70°C. All samples and standards were pretreated with /3-propiolactone prior to assay. Blood and CSF samples were assayed for TNFa by a standard ELISA technique (British Biotechnology, human TNFa Quantikine assay). The minimum detectable level was 4.4 pg/ml in calibrator diluent for CSF and blood. The maximum TNFa level in serum and plasma samples collected from apparently healthy normal donors (n = 40) assayed by the manufacturers was 15.6 pg/ml. 2.3. Statistics The Mann Whitney-U test was used to compare TNFa immunostaining between cords with and without myelopathy. Spearman’s rank coefficient ( rs) was used for correlations between severity of VM and grade of immunostaining for TNFcu. The Kruskall-Wallis one-way analysis of variance was used to compare CSF and blood TNFcv levels in the groups studied. The unpaired t-test was used to compare post mortem delay between patients with and without VM.
3. Results 3.1. Immunocytochemistry for TNFcx in the spinal cord See Tables 1-3, and Figs. 2-4. The main finding was the presence of abundant TNFcv immunostaining in macrophages within the areas of vacuolar change in the cords with VM. The cells staining positive were morphologically macrophages and microglia, although staining was also seen in some endothelial cells. The amount of immunostaining for TNFcr, did not correlate with the pathologic severity of VM (r, = 0.41, p = 0.13); reflecting the observation that TNFcv was present in conspicuous amounts even in mild-moderate VM. Staining for TN& was however, significantly higher in cords with VM compared with HIV-seropositive AIDS controls and HIVseronegative cords without VM (Mann-Whitney U, p = 0.00004). This observation still held (p = O.OOl), if the HIV-seronegative group was excluded, since this group would be expected to have low levels of TNFcx. TNFcv staining was significantly higher in the HIV-seropositive group as a whole compared with the HIV-seronegative cords (p = 0.001). In the cords with VM, the distribution of TNFa staining corresponded with areas of pathology; most of the positive-staining macrophages were situated adjacent to and within vacuoles in the lateral, posterior and anterior columns. In the cases of mild VM, staining was prominent in activated microglia and macrophages in the lateral and posterior columns, despite the presence of relatively few vacuoles. In the HIV-seropositive AIDS control cords, one showed diffuse staining in activated microglia, and 3 showed discrete positivity in occasional microglial cells. The location of the cells in these cords did not correspond to the areas normally involved by VM. In the HIVseronegative controls, staining was only seen in a few microglial cells in one cord, and was markedly less than that seen in the HIV-seropositive cords. In patient 3, spinal cord tissue as well as CSF and blood were available; there was no elevation in blood or CSF TNFa levels despite the strong immunopositivity demonstrated in the cord. 3.2. HIV immunostaining HIV p24 immunostaining was positive in 3/15 spinal cords of patients with VM, and in none of the 4 HIVseropositive AIDS control cords. In all three cords staining positive for HIV, multinucleated giant cells were also present, suggesting the presence of concomitant HIVmyelitis. 3.3. CSF and blood TNFo lecels 3.3.1. Patients. Vacuolar myelopathy (n = 16) TNFa was detectable in the CSF of only one patient with VM. In the blood, the TNFcx was detectable in 12
S.V. Tan et ul./Journal
of theNeurological Sciences 138 (1996) 134-144
patients; median 10.7 pg/ml (range O-292.9 pg/ml). In the 5 patients with post mortem confirmation, TNFa was undetectable in CSF in all, and was detected in the blood in three (37.2, 11.4, 9.3 pg/ml) (Table 4). 3.3.2. HIV-seropositil’e
controls
3.3.2.1. Non-cacuolur myelopathies (n = 8). CSF TNFcY was undetectable in 5 patients. In the remaining 3, the CSF TNFcv levels were 9, 11 and 46 pg/ml. In the blood, TNFu was detectable in 6 patients; median 19.25 pg/ml (range O-673.6 pg/ml). 3.3.2.2. CNS infection without clinical myelopathy (n = 31). CSF TNFcx was undetectable in 21 patients. In the remaining 10, the median CSF TNFL~ was 49.5 pg/ml (range 24.2 to 72.4 pg/ml). In the blood, TNFcv was
141
detectable in 14 patients; median 45.2 pg/ml 352.2 pg/ml).
(range O-
3.3.2.3. No clinical or radiological ecidence of CNS disease fn = 9). CSF TNFa was undetectable in 7 patients. In the remaining 2, the levels were 20.2 and 61.4 pg/ml. In the blood, TNFar was detectable in only 1 patient (9.3 pg/ml). 3.3.3. HIV-seronegatiue controls with motor neurone disease fn = 7) CSF TNFcv was undetectable in 4 patients. In the remaining 3, the levels were 61.4, 64.1 and 77 pg/ml. TNFcv was undetectable in all the blood samples in this group. The results were analysed using the Kruskal-Wallis one-way analysis of variance as the data were not normally
Table 4 CSF and blood levels of TNFa in patients with vacuolar myelopathy and HIV seropositive and HIV seronegative controls Vacuolar myelopathy
HIV seropositive controls Non-vacuolar myelopathies
Pt
Id 2 3 4” 5 6 7” 8 9 10 II I2 I3 14” 15” I6
CSF TNFcx
Blood TNFcx
(pg/mO
(pg/mO
0 0 0 0 69.6 0 0 0 0 0 0 0 0 0 0 0
0 292.9 51.8 31.2 0 0 0 7.5 12.8 II 10.5 12.7 9.7 II.4 9.3 10.9
Pt
CSF TNFcv
Blood TNFa
(pg/ml)
(pg/ml)
I7 18” I9 20 21 22 23 24
46. I 0 0 0 Il.6 0 0 9
673.6 31.2 0 0 10.4 IO 9.1 9.7
HIV seronegative controls CNS infection. No myelopathy
No CNS disease
Pt b
Pt
25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 d 49 50 51 52 53 54 55’
CSF TNF~Y
Blood TNFLV
h/ml)
(pg/ml)
0 0 44.4 0 72.4 0 0 0 0 0 0 0 0 0 0 50.3 54.9 24.2 48.6 46.3 0 0 48.2 53.6 0 0 0 54.5 0 0 0
0 62.5 225.1 0 45.1 0 0 0 15 0 1.5 10.1 IO.9 IO 0 87.8 352.2 45.7 96.7 0 0 0 44.8 0 0 0 0 89 0 0 0
56 57 58 59 60 61 62 63 64
Motor neurone disease
CSF TNFcv
Blood TNFa
(pg/ml)
(pg/ml)
0 0 0 20.2 61.4 0 0 0 0
0 0 0 0 0 0 0 0 9.3
Pt TNFCV 65 66 67 68 69 70 71
CSF
61.4 0 II 64.1 0 ,o 0
Blood
0 0 0 0 0 0 0
Pt = patient; CSF = cerebrospinal fluid; TNFcv = tumor necrosis factor alpha; a Post mortem confirmation of diagnosis; h Clinical diagnosis as follows: Patients 25-39 = encephalitis, 40-46 = cryptococcal meningitis, 47-50 = progressive multifocal leukoencephalopathy, 5 l-52 = cerebra] toxoplasmosis, 53-54 = viral meningitis; 55 = cerebral aspergillosis.
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S.V. Tan et al. / Journal of the Neurological Sciences 138 (19961 134-144
distributed. There was no difference in the CSF TNFa levels (p = 0.25) but blood TNFa levels were significantly different ( p = 0.005) between the groups. The latter were higher in the patients with VM, non-vacuolar myelopathy, and CNS infection without myelopathy compared with the HIV-seropositive patients without CNS disease and HIV-seronegative patients with motor neurone disease. 3.4. Serum 812 and folate
In the pathological series, B12 levels were low in l/4 patients with VM tested. Red cell and serum folate levels were normal. In the clinical series, B12 levels were low in 3/16 patients with VM, and serum folate levels low in l/ 16 patients. Red cell folate levels were normal.
4. Discussion Our results show that TNFo is present in significant amounts in macrophages, microglia and endothelial cells within the areas of pathology in VM. In contrast to Tyor et al (Tyor et al., 1993) who recently reported the presence of TNFa! in spinal cords with VM by immunocytochemistry, we found increased amounts of TNFa in cords with VM compared to cords from HIV-seropositive patients without VM. The amount of TNFo staining did not correlate with the severity of the VM. This may be due to the fact that in mild VM the activated microglia contained conspicuous amounts of TNFcu. These microglia may be the precursors of some of the TNFcr staining macrophages seen in the lesions of more advanced VM (Tan et al., 1995). The presence of abundant TNFcv in mild, presumably early lesions would support the view that it may play a role in the myelin vacuolation seen in VM. Our finding of an increased amount of TNFcv staining in spinal cords from HIV-seropositive patients compared to HIV-seronegative normal controls concurs with the results of Tyor et al. (Tyor et al., 1993). The staining in these cords was mainly in microglia and endothelial cells, and may suggest a background of immune activation within the CNS of HIV-seropositive patients. It is also possible that some of these cords may represent the very early stages of VM. The presence of TNFcv within the spinal cord of patients with VM was not reflected by increased levels of TNFcv in the CSF. Similarly increased TNFcr in brain tissue has been found in multiple sclerosis (Selmaj et al., 1991) though in CSF it has been reported as both absent (Gallo et al., 1989) and elevated (Sharief and Hentges, 1991). TNFo has a short half-life in biological fluids (Selby et al., 1987). CSF TNFa levels could therefore be expected to be mildly, or only modestly, elevated in conditions where it is produced largely within the
parenchyma of the CNS itself rather than in the meninges. In our patients, as in a previous report (Mastroianni et al., 1992) TNFa was most frequently detected in the CSF of patients with cryptococcal meningitis (5/7). The higher blood levels of TNFa in the patients with VM, with non-vacuolar myelopathy, and those with CNS disease, compared with HIV-seropositive patients without CNS disease and with HIV-seronegative patients with motor neurone disease may suggest a concomitant systemic immune response in the former conditions. The proportion of patients with AIDS in the HIV-seropositive without CNS disease group was similar to the other groups. Thus, although a general immune activation might be expected in patients with advanced AIDS it would not, by itself, account for the elevated blood TNFa levels found in the former groups. The group of patients with MND were older than the other groups; however, TNFo levels have not been shown to decrease significantly with age (Riancho et al., 1994). Our results suggest that locally produced TNFa may play a role in the myelin damage and vacuolation seen in VM. Its presence in the lateral and posterior columns in mild, presumably early, VM and its capacity to produce ballooning of myelin in cultures of mouse spinal cord tissue (Selmaj and Raine, 1988) suggest that TNFcv may be directly involved in the pathogenesis of the vacuolar change. It is not known why myelin vacuolation occurs in such a distinctive distribution within the thoracic cord. The processes associated with chronic macrophage activation and myelin damage within the spinal cord may have some bearing on the anatomic distribution of the pathological change in VM. One hypothesis is that in the late stages of HIV infection, immune activation within the CNS, either due to the presence of HIV encephalitis or opportunistic infections, may lead to the local production of myelin/membrane damaging cytokines such as TNFa (Tyor et al., 1993) toxins, or oxygen radicals by microglia, resident macrophages and endothelial cells. Damaged or degenerating myelin fibres may act to attract scavenging macrophages. Those macrophages expressing gp120 may further damage myelin by binding to sulphatide and myelin associated glycoprotein on oligodendrocyte membranes (van den Berg et al., 1992). The local presence of TNFa may result in an upregulation of MHC Class II antigen expression on microglia and macrophages (Sherry and Cerami, 1988). These cells, having ingested and processed myelin protein, may then act as antigen presenting cells thus further enhancing the immune reaction. Such chronic myelin damage is likely to lead to increased demands for metabolic products required for repair. Critical to these repair mechanisms is the methylation of fatty acids, phospholipids, polysaccharides and proteins, such as myelin basic protein, by the body’s universal methyl donor (S-adenosylmethionine), a product of the methyl transfer pathway involving B 12 and folate metabolism (Surtees and Hyland, 1990a). As this is the
S.V. Tan et al. /Journal
of the Neurological Sciences 138 i 19961 134- 144
sole pathway for the generation of methyl groups, should the consumption of methyl groups for repair mechanisms become excessive, a secondary methyl group deficiency similar to that seen in vitamin B 12 and folic acid de% ciency may develop (Surtees et al., 1990b), thus predisposing the spinal cord to myelin degeneration similar to that seen in subacute combined degeneration of the cord (SACD)(Hyland et al., 1988). Such a cellular deficiency may account for the close histological resemblance of VM to SACD without a demonstrable association with reduced serum B12 and folate levels, a finding we have also confirmed. Further myelin degeneration may attract more macrophages and perpetuate the process.
Acknowledgements This work was supported by the North West Thames Regional Health Authority and the Special Trustees of the Westminster and Roehampton Hospitals. We thank Professor F Scaravilli from the Institute of Neurology for his advice and for providing facilities for the immunocytochemistry. We are also grateful to Dr A. Ciardi and Mr A. Beckett for technical help, and to Dr J.N. Harcourt-Webster and Dr S. Lucas for performing the post mortem examinations.
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