Autoantibodies in Neurodegenerative Diseases: Antigen-Specific Frequencies and Intrathecal Analysis

Neurobiology of Aging, Vol. 19, No. 3, pp. 205–216, 1998 Copyright © 1998 Elsevier Science Inc. Printed in the USA. All rights reserved 0197-4580/98 $19.00 1 .00

PII:S0197-4580(98)00049-9

Autoantibodies in Neurodegenerative Diseases: Antigen-Specific Frequencies and Intrathecal Analysis J. W. TERRYBERRY,1 G. THOR, AND J. B. PETER Specialty Laboratories, 2211 Michigan Avenue, Santa Monica, CA 90404 Received May 6, 1997; Revised January 22, 1998; Accepted January 29, 1998 TERRYBERRY, J. W., G. THOR, AND J. B. PETER. Autoantibodies in neurodegenerative diseases: antigen-specific frequencies and intrathecal analysis. NEUROBIOL AGING 19(3) 205–216, 1998.—The frequency of autoantibodies (AAbs) was surveyed in several neurodegenerative diseases, other neurological diseases, and controls using antigen-specific EIAs for neurofilament heavy subunit, tubulin, glial fibrillary acidic protein, S100 protein, tau, b-amyloid peptide, myelin basic protein, and heparan sulfate proteoglycan. High frequencies of sera and cerebrospinal fluid tubulin AAbs were found in Alzheimer disease (62% and 69%, respectively), Parkinson disease (27% and 70%), amyotrophic lateral sclerosis (54% and 67%), and in sera from multiple sclerosis (50% and 67%), optic neuritis (85%), Guillain-Barre´ syndrome (88%), and vascular dementia (52%). High frequencies of neurofilament heavy subunit AAbs were detected in Guillain-Barre´ syndrome, chronic peripheral neuropathy (88%) and optic neuritis (62%); whereas, some Alzheimer’s disease (33%) and vascular dementia (44%) patients had glial fibrillary acidic protein AAbs. Lower frequencies of other AAbs were found in patient groups. AAb results were also compared to functional assessment of blood-brain barrier integrity in Parkinson’s disease and Alzheimer’s disease. The relevance of these AAbs to pathogenesis and/or course of neurologic diseases merits further study with particular reference to subgrouping and prognosis. © 1998 Elsevier Science Inc. Alzheimer disease Parkinson disease Amyotrophic lateral sclerosis Autoantibodies Neurofilament Tubulin

Multiple sclerosis

Neuropathy

pathologic state of immune activation, which includes autoimmunity and inflammation involving lymphocytes, macrophages, microglia, and complement pathways, in a putative subset of AD patients (2,37,52,56,58,59,78,103,109,110,123,124,128,131). Brains from patients with Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) also demonstrate elevated expression of HLA-DR, M-CSF, C3, and C4 (3,7,32,38). Tissue and cellular targets of AAbs variably associated with AD and other NDDs include microglial cells, astrocytes, cholinergic neurons, vasculature, and pituitary cells (11,14,22,36,39,47,50,65,67,70,76,82,88, 91,108,117). Moreover, immunoglobulin G (IgG) and complement have been detected in neurofibrillary tangles (NFTs) and senile plaques (SPs) (primarily core rather than diffuse SP) (30,47,55,65, 72,92); although their presence in NFTs and possible role in SP pathology remain unclear (31). However, AD plaques demonstrate immunoreactivity for the IgG receptors FcgRI, II, and III. SPassociated microglia show elevated expression of MHC class II, and most all plaques are HLA-DR positive (138 –140) Immunoglobulin deposits are also detectable in ischemic lesions after CNS infarction (122). Known autoantigens in NDDs include mostly cytoskeletal and intermediate filament components such as neurofilament (NF)

ABERRANT immune functions are well recognized in heterogenous neurodegenerative diseases (NDDs). These include abnormalities of both cellular and humoral immunity and inflammatory responses (28,32,44,51,77,95,104,106,107). In the aged brain, increased central nervous system (CNS) activation of microglia and macrophages is seen with corresponding increases in the expression of complement 3 receptor (C3R), major histocompatibility complex (MHC) class II, CD4, and leukocyte common antigen (LCA) (89). Aged lymphocytes demonstrate altered T suppressor function, impairments of leukocyte inhibitory factor (LIF), and lymphocyte-derived chemotactic factor, and altered proliferation responses (5,87). Immunosenescence is characterized by elevated levels of interleukin (IL)-6 and IL-10, and age-related increases in autoantibody (AAb) production and CD51 B1 cells are also well documented (40,105,137). In Alzheimer disease (AD), increased brain and/or serologic concentrations of CD41CD45- and CD81 T cells, as well as HLA-DP, -DQ, and -DR; LCA, MHC class II, Fcg receptor, IL-1, -2 and -6, tumor necrosis factor (TNF), IL-2 receptor, CD45, soluble CD8, membrane attack complex (MAC), C1q, reacted C1 inhibitor, clusterin (apoJ), circulating immune complexes (CIC), and macrophage colony stimulating factor (M-CSF) all indicate a potentially

1 Address correspondence to: Jeff W. Terryberry, Specialty Laboratories, 2211 Michigan Avenue, Santa Monica, CA 90404; E-mail: [email protected].

205

206

TERRYBERRY ET AL.

(10,16,34,63,115), glial fibrillary acidic protein (GFAP) (83,118, 119), vimentin (102), and tubulin (17,62,96), as well as S100 protein (60). AD, which is pathologically characterized by the CNS deposition of amyloid in SPs, is also accompanied by formation of AAbs to b-amyloid peptide (BAP) (35,46,55,85). Other immune functionalities of BAP, a potential acute phase reaction product of inflammation (54), include activation of the classical complement pathway and microglial cells (37,59,95,131). This study, the largest and most comprehensive of its kind to date, evaluates the frequency of eight AAbs using highly specific and optimized EIAs, and further elaborates the significance of autoimmunity in several NDDs including, AD, vascular dementia (VD), PD, and ALS, as well as several inflammatory demyelinating disease controls such as multiple sclerosis (MS), optic neuritis (ON), Guillain-Barre´ syndrome (GBS), and chronic inflammatory demyelinating polyradiculoneuropathy (CIDP), as well as in normal controls and other neurologic diseases (OND). AAb levels examined include those immunoreactive with GFAP, NF-heavy (H) subunit, S100 protein, tau, BAP(1– 42), tubulin, myelin basic protein (MBP), and heparan sulfate proteoglycan (HSPG). PD and AD samples were also used to evaluate blood-brain barrier (BBB) integrity and intrathecal AAb production. MATERIALS AND METHODS

Patient and Control Samples 1. Twenty-six definite AD (postmortem confirmed) antemortem, non-lipemic sera (marble-top Vacutainer serum tube; BectonDickinson, Franklin Lakes, NJ) and 47 AD postmortem, non-hemolyzed cerebrospinal fluid (CSF) (postmortem interval (PMI) , 12 h) inclusive of 12 paired CSF and sera samples [from Dr. T. Pallidino, University of California, San Diego, CA; and the National Neurological Research Specimen Bank (NNRSB), Veterans Administration Medical Center (VAMC), Los Angeles, CA, which is sponsored jointly by NINDS/NIMH, National Multiple Sclerosis Society, Hereditary Disease Foundation, Comprehensive Epilepsy Program, Tourette Syndrome Association, Dystonia Medical Research Foundation and Veterans Health Services and Research Administration, Department of Veterans Affairs]. 2. Twenty-five PD antemortem, non-lipemic sera and 16 PD postmortem, non-hemolyzed CSF (PMI , 12 hr.) inclusive of 11 paired CSF and sera [from Dr. S. Jacobson, National Institutes of Health (NIH), Bethesda, MD; and NNRSBVAMC]. 3. Twenty-six ALS antemortem sera and 3 ALS antemortem (lumbar puncture) CSF inclusive of 2 matched CSF and sera [from Dr. D. Lacomis, University of Pittsburgh, PA]. 4. Forty-six relapsing-remitting MS (RR-MS) sera and 17 CSF inclusive of 17 paired CSF and sera; and 42 chronic-progressive MS (CP-MS) sera and 24 CSF inclusive of 24 paired CSF and sera [from Dr. S. Jacobson, NIH; and Dr. R. Baumhefner, University of California, Los Angeles, CA]. 5. Thirty-five ON sera and 30 ON CSF inclusive of 30 paired CSF and sera [from NNRSB-VAMC]. 6. Twenty-five VD sera with Mini Mental Status Exam (MMSE) scores ,28 (DSM III-R dementia criterion) [from Dr. M. Fisher, University of Southern California, Los Angeles, CA]. 7. Ten OND sera, including 2 Pick disease, 3 astrocytoma, 1 myelopathy, 1 stiff-man syndrome, 1 meningitis, 1 normal pressure hydrocephalus (NPH), and 1 Graves disease; and 6 paired CSF (1 Pick disease, 3 astrocytoma, 1 myelopathy, and 1 meningitis) [from Dr. F. Gaskin, University of Virginia; NNRSB-VAMC; and Dr. A. Bradwell, Birmingham University, UK; and Binding Site, Inc. (BSI), San Diego, CA].

8. Twenty-four GBS sera [from Dr. A. Bradwell, BSI]. 9. Nine CIDP and 8 sensorimotor neuropathy (SMN) sera [from Dr. A. Bradwell, BSI]. 10. Thirty anti-neuronal AAb negative control CSF (nonreactive to neuroblastoma surface antigens by flow cytometry) [from Dr. P. D’Amore, Specialty Laboratories, Santa Monica, CA]. 11. Forty-four normal human sera [from Red Cross, Los Angeles, CA]. All other specimens (4 –11 above) were obtained from living patients, and all sera and CSF were shipped frozen, then thawed and aliquoted, and stored at 220°C. Single aliquots were thawed once more for use in the AAb EIAs. Antigens and Antibodies NF-H (200 kDa) (bovine spinal cord); GFAP (52 kDa) (bovine spinal cord); and S100 protein (human brain) were obtained from Biodesign Intl. (Kennebunk, ME). HSPG (bovine intestinal mucosa), BAP(1– 42) (synthetic), tau (bovine brain), tubulin (bovine brain), and MBP (bovine brain) were obtained from Sigma Biosciences (St. Louis, MO). The following Abs were used in EIA and immunoblot for standards and controls: mouse anti-rat b-tubulin MAb 380 (Chemicon Intl, Inc., Temecula, CA); anti-GFAP MAb SMI 26 (Sternberger Monoclonals, Inc., Baltimore, MD); anti-NF-H MAb SMI 35 (phosphate-dependent) (Sternberger Monoclonals, Inc.); mouse anti-human GFAP MAb 6F2 (Biodesign Intl.); rabbit anti-chick tau polyclonal (Accurate Chemical and Scientific Corp., Westbury, NY); goat anti-bovine S100 polyclonal (Calbiochem, La Jolla, CA); mouse anti-bovine tau MAb (Calbiochem); rabbit anti-BAP(1240) polyclonal (Sigma Biosciences, St. Louis, MO); rabbit anti-NF-H(200) polyclonal (Sigma Biosciences); rabbit anti-human MBP polyclonal (Chemicon Intl.); rabbit anti-human MBP polyclonal #201 (a generous gift from Dr S.G. Li; Specialty Laboratories); rabbit anti-BAP(25235) polyclonal (Boehringer Mannheim Biochemica, Indianapolis, IN); mouse anti-b tubulin MAb KMX-1 (Boehringer Mannheim); human anti-human BAP MAb MRE 148 (a generous gift from Dr. Felicia Gaskin, University of Virginia, Charlottesville, VA); and mouse anti-HSPG MAb 45924F2 (Chemicon Intl.). Each mono- and polyclonal antibody used in the immunoblot and EIA assays demonstrated interspecies (bovine and human) antigen crossreactivity. For example, the Sigma and Boehringer anti-BAP polyclonals both recognized BAP(1– 42) antigen, as did the human anti-human BAP MAb MRE 148 (46). EIA Protocol The EIA standard protocol was previously described (120). In brief, Immulon 1 (Dynatech; Chantilly, VA) 96-well microtiter plates were coated with 250 ng/well antigen in carbonate buffer, pH 9.6, overnight at 4°C. An exception to this was tubulin, which was coated at 1.0 ug/well. Higher signal-to-noise ratios were obtained using Immulon 2 plates for MBP AAb assays (data not shown). After coating, plates were washed 13 with phosphate buffered saline (PBS) (50 mM sodium phosphate, pH 7.4 containing 150 mM NaCl) plus 0.05% Tween-20 (PBST), and then blocked with PBS plus 1% bovine serum albumin (BSA) (BSA; fraction V; Sigma Biosciences) assay diluent for 30 min at room temperature (RT) with shaking. After blocking, plates were washed 13 with PBST and then 100 mL of appropriately diluted standards, controls, and patient samples (1:300 human sera and undiluted CSF) were pipetted into corresponding wells and incubated 1 h at RT with shaking. Plates were then washed 33 with PBST, or in the case of MBP AAbs, with PBS plus 0.05% Micro

AUTOANTIBODIES IN NEURODEGENERATION zwitterionic detergent (PBSM) (International Products Corp.; Burlington, NJ); and goat anti-mouse, -rabbit, or -human IgG (and IgM for human AAbs only) conjugated to alkaline phosphatase (American Qualex; La Mirada, CA) were added at 1:100024000 dilutions in assay diluent to appropriate standard, control, or sample wells (100 uL). After incubation for 1 h at RT with shaking, plates were washed 33 and then developed with 1 mg/mL paranitrophenyl phosphate (pNPP) and the absorbance determined at 405 nm on a plate reader (Molecular Devices; Menlo Park, CA) equipped with data reduction software (Softmax). For determination of MBP and tubulin AAbs in MS and ON CSF, samples were acid-dissociated prior to the assay by adding 5 uL 1N HCl to 300 uL CSF, incubating for 30 min. at RT and then neutralizing with 5 uL 1N NaOH. Neutralized, acid-dissociated CSF was then used immediately for the assays. Antigen Isolation from Hippocampus Aqueous, nonionic detergent-solubilized and ionic detergentsolubilized hippocampal extracts from AD and age-matched control brains were used for immunoblot analyses. In brief, frozen brains were partially thawed and the hippocampus and adjacent temporal cortical structures removed and placed in 30 mL of ice-cold 50 mM Tris-base, pH 8.0, containing 125 mM NaCl, 1 mM PMSF, and 1 ug/mL each of the following protease inhibitors: leupeptin, antipain, pepstatin A, and aprotinin (Sigma Biosciences). Hippocampi were then homogenized using a Polytron homogenizer, followed by rehomogenization with a Dounce homogenizer to a uniform consistency. Homogenates were then centrifuged for 15 min. at 4,000 rpm, 4°C, and then for 1 h at 45,000 rpm, 4°C. The resulting supernatant (SN1) was diluted to 3 mg/mL and aliquoted for storage at 270°C. The pellets from the above two centrifugations were combined, rehomogenized (Dounce), and solubilized with the above buffer plus 1% Nonidet P240 for 2 h at 4°C. SN2 was obtained after centrifugation as above, adjusted to 3 mg/mL, and aliquoted for storage at 270°C. The second set of pellets were rehomogenized and solubilized with 1% sodium dodecylsulfate (SDS) overnight at 4°C and then centrifuged to yield as above SN3, which was also adjusted to 3 mg/mL and stored in aliquots at 270°C. Immunoblotting SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was performed as described (69) using 4220% Tris-glycine MiniProtean II gels (Bio-Rad, Hercules, CA). Purified antigens were run at 125 ug/lane and AD hippocampal extracts (SN123) at 120 ug protein/lane along with high and low prestained molecular weight markers. After standard transfer (126) to 0.22 um nitrocellulose sheets, the immunoblots were probed with mono- or polyclonal antisera and human NDD sera for confirmation of AAb immunoreactivity. Calculations The following CSF analysis parameters were determined for paired CSF and serum samples in AD and PD. Autoantigen-specific antibody indices (ASAI) (90). ASAI is a measurement of the ratio of antigen-specific to total Ig in CSF compared to the ratio in serum and is calculated by: ASAI 5

[ [

]

Antigen-specific Ig in CSF 4 Total Ig in CSF

]

Antigen-specific Ig in Serum Total Ig in Serum

207 An index $2 is strong evdience that intrathecal synthesis of autoantigen-specific Ig is occuring in the CNS. IgG Synthesis (IgG synth), Albumin quotient (Q Alb) and IgG(loc) (93). The Tourtellote formula (125) was used to calculated IgG synth as: IgG synth rate (mg/24 h) 5 {[CSF IgG 2 (S IgG/369)] 2 [(CSF Alb 2 (S Alb/230)) 3 (S IgG/S Alb) 3 0.43]} 3 5 Values .4.2 are indicative of intrathecal synthesis in patients with intact BBB. Q Alb 5 ( Albumin in CSF/Albumin in serum) Values . 7 are indicative of disturbed BBB permeability (134). Because alterations in BBB permeability that give rise to elevated Q Alb values can give spuriously elevated IgG synth values (90), the local intrathecal production of IgG (93) was calculated as: IgG Loc 5 [QIgG 2 QLim(IgG)] 3 IgG(serum) where QIgG 5 CSF IgG/Ser IgG, and QLim(IgG) is the upper limit reference range equal to: QLim(IgG) 5 0.93 [ Î

QAlb2 1 6 3 1026] 2 1.7 3 1023

Values . 2.6 are abnormal (mean 1 2 standard deviations (SD) of normals) RESULTS

Each optimized EIA had intra- and interassay coefficients of variation ,15%. Normal distributions of control human sera were demonstrated at serial dilutions; clinical cut-off values were the mean plus 3 SD of the mean optical density (OD) of the normals. Dilutions for each serum AAb assay were determined as the dilution yielding a mean OD ,0.100 for the normal controls. In all control sera, a 1:300 serum dilution fulfilled these criteria and was thus used to screen pathological specimens. Each patient sample was quantified based on a standard curve of Ag-specific mono- or polyclonal antisera; wherein, standard 1 yielded an OD of 1.0 and a value of 200 EIA Units within 20 min. of development time. Antibodies used for standard curves at appropriate serial dilutions were: anti-rat b-tubulin MAb 380; anti-GFAP MAb SMI 26; goat anti-bovine S100 polyclonal; anti-NF-H MAb SMI 35; rabbit anti-BAP (1– 40) polyclonal; anti-bovine tau MAb; rabbit antihuman MBP polyclonal 201; and anti-HSPG MAb 459 – 4F2. A similar analysis was performed to determine the control CSF distribution: neat, undiluted CSF gave .95% OD values ,0.100 and undiluted CSF was thus used to measure AAbs in pathological specimens with EIA Unit values generated from a standard curve. Using an assay cut-off value of 10 EIA Units (;0.150 OD), tubulin AAbs were found in 0/44 normal human sera (NHS) controls, 4.5% (1/22) of control CSF samples, 17% (1/6) of CP-MS CSF, 39% (7/18) of CP-MS sera, 23.5% (4/17) of CIDP/SMN sera, 20% (4/20) of PD sera, and 50290% of all other patient sample groups (AD, ALS, VD, RR-MS, ON, and GBS) excluding the miscellaneous OND group (Table 1). No other AAbs were found in control CSF; whereas, NHS contained 2.0% (1/44) AAbs to NF-H, 4.5% (2/44) to GFAP and 4.5% (2/44) to tau. AD sera showed the highest frequency of AAbs to tubulin (62%), followed by GFAP (33%), NF-H (29%), BAP (23%), and HSPG (23%); AD CSF showed a similar pattern of autoreactivity. Remaining patient group AAb frequencies are listed in Table 1. AAb multireactivity in AD and PD sera and CSF and in ALS and VD sera showed that 86% of AD sera, 76% of AD CSF, 62% of

208

TERRYBERRY ET AL. TABLE 1 FREQUENCY OF AUTOANTIBODIES IN PATIENT AND CONTROL GROUPS Patient Group AD

Autoantigen

PD

ALS

RR-MS

Sera

CSF

Sera

CSF

Sera

CSF

Sera

CSF*

62% (13/21) 33% (7/21) 29% (6/21) 23% (6/26) 0

69.0% (20/29) 23% (11/47) 14% (4/29) 10% (3/29) ND

20% (4/20) 0

60.0% (9/15) 13% (2/16) 31% (5/16) 13.0% (2/16) ND

67% (2/3) 0

50.0% (5/10) 0

67% (4/6) ND

0

ND

ND

22% (8/37) 23% (3/13) 25.0% (9/36) ND

ND

62.5% (10/16) ND

S100

23% (5/22) 0

ND

0

ND

Tau

0

54% (14/26) 19% (5/26) 15% (4/26) 9.0% (2/26) 4.0% (1/26) 24.0% (5/21) 9.0% (2/26) 4.0% (1/26)

ND

0

ND

Tubulin GFAP NF-H BAP(1-42) MBP HSPG

3% (1/37) 0

33% (7/21) 14% (3/21) 15.0% (3/20) 13% (2/16) 0 0

ND 0 0

ND 0

ND

*, with acid dissociation for MBP and tubulin Abs (only). Abbreviations: AD, Alzheimer disease; Ag, antigen; ALS, amyotrophic lateral sclerosis; ANeA, antineuronal autoantibody; BAP, beta-Amyloid peptide; CIDP, chronic inflammatory demyelinating polyradiculoneuropathy; CP-MS, chronic-progressive multiple sclerosis; CSF, cerebrospinal fluid; GBS, Guillain-Barre´ syndrome; GFAP, glial fibrillary acidic protein; HSPG, heparan sulfate proteoglycan; MBP, myelin basic protein; ND, not determined; NH, normal human; ON, optic neuritis; OND, other neurologic disease; PD, Parkinson disease; RR-MS, relapsing-remitting multiple sclerosis; SMN, sensorimotor neuropathy; and VD, vascular dementia.

PD sera, 69% of PD CSF, 77% of ALS sera, and 68% of VD sera contained at least one AAb (Table 2). In Tables 3 and 4, AAb reactivities in paired sera and CSF from AD and PD patients were compared for the total number of sera and CSF AAbs and immunoglobulin isotype concentrations. Correlation coefficients indicate that the total number of AD serum and CSF AAbs was associated with the levels of IgM (r 5 0.45); little association was seen between the number of AAbs in sera and the number of AAbs in CSF in AD (r 5 0.35) (Table 3b). In contrast, the number of AAbs in CSF and the number in sera were more tightly correlated in PD (r 5 0.65). Furthermore, the number of CSF AAbs in PD was slightly associated with CNS IgG synth results (r 5 0.49) and CSF IgG levels (r 5 0.42), but not CSF IgM levels (Table 4b). In AD, the number of sera AAbs was not correlated to sera concentrations of IgG or IgA (Table 3b); whereas, in PD, the number of sera AAbs was very much associated with the sera levels of IgG (r 5 0.99). BBB integrity was assessed, and correlated for the first time to NDD-related autoimmunity by calculation of intrathecal synthesis of IgG (IgG synth, Tourtellotte formula) (125), the Q Alb, IgG(loc) (93), and the ASAI (90). Sixty-four percent of 11 matched sera and CSF from PD patients had CNS IgG synth values . 4.2; whereas, 33.3% of 12 matched AD samples had values . 4.2 for this parameter. Standard assessment of BBB damage by the albumin quotient (Q Alb), revealed that 55% of matched PD and only 25% of matched AD samples demonstrated BBB damage (Tables 3a and 4a). All patients with elevated Q Alb values also had elevated IgG synthesis values. Adjustment for BBB leakage, which can give a spuriously elevated IgG synth value, by the evaluation of local IgG production revealed that 0% AD and 27% of PD paired specimens had abnormal IgG(loc) values (Tables 3a and 4a). The evaluation of intrathecal AAb production was further assessed by calculating the

ASAI; 2/12 (17%) of AD and 2/11 (18%) of PD matched samples demonstrated intrathecal production of tubulin AAbs only (Table 5). No correlations between ASAI and IgG(loc) values were seen in either AD or PD (Tables 3b, 4b, and 5). DISCUSSION

This study confirms, consolidates, and extends previous demonstrations of specific AAbs in NDD populations (63,83,115,121), and correlates autoreactivity results to BBB functional parameters, while focusing on a specific panel of defined neuronal autoantigens. S100, in contrast to a previous report (60) and tau were shown to be extremely poor autoantigens; only 1 AD CSF and 2 ALS sera contained S100 AAbs; and only 1 ALS patient serum had a detectable anti-t response. No previous studies reported on the human humoral immune response to tau, which is an integral component of NDD NFTs (57,100). However, our results do not rule out the possibility that a paired helical filament (PHF)-t epitope specific to AD may be autoantigenic. Unexpectedly, tubulin AAbs were prevalent in most sera: 62% AD, 52% ALS, 39250% MS, 52% VD, and 88% GBS but not in normal controls (4.5%) and in only 24% of SMN/CIDP (Table 1). Thus, tubulin AAbs in both sera and CSF are a common neurodegenerative pathological event. In aged rodent brain, there is a decrease in the slow axonal transport rate of both tubulin and NF-H (80). Axonal transport is also impaired by antisera to purified pig brain tubulin, although the cellular uptake of such antibodies and their mechanism of transport inhibition is unclear (61). Moreover, tubulin is a probable component of NFTs (53,92). A few reports are available on the detection of tubulin AAbs in GBS and CIDP (17,71), and ALS (18); insulin-dependent diabetes mellitus (IDDM) with and without neuropathy (20,96); experimental thymic cytotoxicity (62); systemic lupus erythematosus (SLE)

AUTOANTIBODIES IN NEURODEGENERATION

209 TABLE 1 CONTINUED

Patient Groups CP-MS

ON

Control Groups

OND

GBS

SMN/CIDP

VD

NH

ANeA Neg.

Sera

CSF*

Sera

CSF*

Sera

CSF

Sera

Sera

Sera

Sera

CSF

39% (7/18) 0

16.6% (1/6) ND

85% (30/35) ND

54% (13/25) ND

ND

ND 0

26% (10/39) 27% (4/15) 27.0% (10/37) ND

ND

ND

60.0% (6/10) ND

0 ND

40.0% (12/30) ND

0

ND

ND

ND

88% (21/24) 21% (5/24) 8% (2/24) ND

0

ND

ND

ND

0

0

0

0

52.0% (13/25) 44.0% (11/25) 24.0% (6/25) 24.0% (6/25) 36.0% (9/25) 17% (4/24) ND

4.5% (1/22) 0 (0/30) 0

46% (11/34) ND

62% (21/34) 20.0% (7/35) 18% (6/34) ND

24% (4/17) 6% (1/17) 88% (15/17) 12% (2/17) 0

0

0

88% (21/24) 0

0

ND

ND

ND

0

0

0

0

ND

ND

ND

(20); paraneoplastic cerebellar degeneration (PCD) [Terryberry JW et al., Submitted]; and MS (74). In addition, sera from patients with leishmaniasis were shown to contain CIC that bound tubulin (99). Our results agree with the frequency (;25%) of tubulin AAbs previously reported in CIDP (71); however, a much higher frequency of autoreactivity was seen in the GBS population examined here. NF-H AAbs are described in several NDDs and ONDs, including: Creutzfeldt-Jakob disease and Kuru spongiform encephalopathies (9,114,121), neuropsychiatric (NP)-SLE (68,94), CNS lesions (23), retinitis pigmentosa and paraneoplastic retinopathy (45,66), thymic epithelial tumors associated with Graves disease (73), and Japanese encephalitis (26), as well as NDDs including PD (33,63), ALS (6), AD (10,16,63,115), and VD (115). NF-H AAbs are known to be directed against phosphate-dependent epitopes (13), and some phosphorylation-dependent immunoreactivity was seen in this study by immunoblot analysis (Fig. 1).

0

4.5% (2/44) 2.0% (1/44) 0 0

0

0

ND

0

0

4.5% (2/44)

0

However, unequivocal NF-H phospho-epitope dependence was not determined due to the polyclonality of the AD sera, and the occurrence of NF-H phosphorylation microheterogeneity (115). NF-H is a component of NFTs in AD and Pick disease (24) and of spinal spheroids in ALS (19,101). Its overexpression and hyperphosphorylation are linked to aberrant axonal transport, perikaryal deposition, and dystrophic neuritogenesis (21,22). In addition, NF-H C-terminus phosphorylation controls its interaction with the cytoskeleton, including tubulin (84). More detailed studies are needed to determine the epitope fine specificity of NF-H AAbs, their affect, if any, on NFT formation, and their potential role in AD central cholinergic neuron pathology (16,39,76). Sera levels of AAbs to tubulin and NF-H were highly correlated in GBS (r 5 0.90) and in ON (r 5 0.78). NF-H AAbs were found at very high frequencies in GBS and CIDP/SMN (88% in both). Our previous study (120) of antimyelin AAb responses in GBS indicated that no single myelin glycolipid was the primary target; maximum AAb

TABLE 2 MULTIREACTIVITY OF NDD SERA AND CSF

# of AAbs

0 1 2 3 4 5 6 Total Autoreactivity

0

ALS Patient AAb Summary

VD Patient AAb Summary

CSF (n 5 16)

Sera

Sera

8 9 4 0

5 7 2 2 0

6 14 3 3 0

62.0% (13/21)

68.8% (11/16)

76.9% (20/26)

8 5 4 1 4 3 0 68.0% (16/25)

AD Patient AAb Summary

PD Patient AAb Summary

Sera (n 5 21)

CSF (n 5 29)

Sera (n 5 21)

3 8 7 3 0

7 17 3 2 0

85.7% (18/21)

75.9% (22/29)

210

TERRYBERRY ET AL. TABLE 3A SUMMARY OF AD PAIRED SERA AND CSF

Patient #

# CSF AAbs

# Sera AAbs

CSF IgG**

Sera IgG**

IgG Synth

IgG(loc)

CSF Alb*

Sera Alb**

Q Alb

CSF IgM*

CSF IgA*

Sera IgM*

Sera IgA*

1 2 3 4 5 6 7 8 9 10 11 12 % Abnormal

1 1 0 0 0 1 1 0 1 1 0 3 58.3

3 2 2 2 1 2 1 1 1 3 0 2 91.7

1.2 1.2 3.0 3.1 2.5 8.0 7.6 10.6 3.0 7.2 5.0 5.4 33.3

1.8 1.5 2.6 0.9 1.2 3.2 1.2 2.3 1.5 1.8 1.5 1.1 41.7

22.2 23.1 1.3 3.8 21.4 211.1 9.5 23.3 21.6 7.1 2.4 1.9 33.3

21.62 22.34 0.414 21.88 23.29 0.420 20.612 27.25 20.882 1.08 21.60 24.61 0

4.2 5.2 9.2 16 13.2 12.4 45.2 44.6 14.2 21.4 19.2 33.0 25

2.5 2.2 4.4 2.9 2.6 2.2 5.0 3.4 4.5 4.1 3.6 3.3 0

1.68 2.36 2.09 5.52 5.08 5.64 9.04 13.12 3.16 5.22 5.33 10.0 25

0.03 0.05 0.03 0.20 0.10 0.08 0.03 0.12 0.02 0.04 0.08 0.31 16.7

0.14 0.23 0.18 0.79 0.19 0.30 0.23 1.24 0.19 0.54 0.28 0.71 25

109.90 140.90 309.50 218.60 108.40 105.70 150.50 103.70 201.20 684.90 151.60 123.90 16.7

334.50 286.60 357.70 919.40 255.70 232.40 239.40 656.80 535.80 691.40 375.40 208.20 58.3

* mg/dL ** g/dL

frequencies were seen to sulfatide and cardiolipin (43249%). The current study suggests that cholinergic axons which are NF-Hpositive (16,39), and not the Schwann cell which does not contain NF, might be the primary AAb targets in peripheral demyelinative neuropathies. Whether anti-NF-H AAbs correlate to axonopathy in peripheral neuropathy also warrants further investigation (18,73). Because 62% of ON sera were NF-H AAb-positive, NF-H might be an important autoantigen in ON. General cholinergic neuron loss with associated Ag shedding might explain the anti-NF-H humoral immune response in VD as well. The NF-H AAb frequencies reported here for AD and VD are lower than a previous study of AD and VD that used phosphorylated NF-H from bovine ventral horn, which was shown to be more autoreactive than that obtained from the dorsal horn of bovine spinal cord (115). The NF-H Ag preparation used in our study was from the entire spinal cord (bovine). The astroglial intermediate filament, GFAP, which is upregulated in gliotic hypertrophy and is also a component of NDDrelated glial fibrillary tangles and glial cytoplasmic inclusions (57,100,122), showed a more restricted pattern of autoreactivity. Only 33% of AD sera demonstrated AAbs to GFAP, as did 44% of VD sera, 19% of ALS sera, and 1 CIDP patient; no other patient sera groups had detectable levels of GFAP AAbs, although 2/11 PD CSF samples were GFAP AAb-positive. Whether sera GFAP AAbs are specific for gliotic reactions in AD and VD is unknown. Further surveys for GFAP AAbs should include patients with

progressive supranuclear palsy and corticobasal degeneration, diseases which also show astroglial histopathology (100). Our results on the frequency of GFAP AAbs in AD and VD validate the GFAP AAb levels in senile dementia (SD) and VD (83), but not at the very high frequencies reported elsewhere (118,119). GFAP AAb frequencies also correlate with the frequency of anti-microglial AAbs reported in AD (21,81). Because GFAP is upregulated during inflammatory responses of microglia (122), these AAbs may provide a link between inflammation and autoimmunity in NDD pathogenesis (28,133). This might be supported by the finding that AAbs in AD were predominantly IgM (Table 3b), which may indicate an acute humoral immune response. BAP(1– 42) was a relatively poor autoantigen. Positive results for BAP AAbs were found in about 25% of AD sera. In addition, 20225% of VD, ON, and both MS groups contained AAbs to BAP. This is not surprising, because the amyloid precursor protein (APP) was recently identified as a component of MS CNS lesions (plaques), while BAP is an integral component of senile plaques in AD and of vascular deposits in congophilic (cerebral) amyloid angiopathy (48,54,79). PD and ALS showed minimal BAP autoreactivities. Investigations are currently in process to determine the autoreactivity of the various soluble APP isoforms. IFN-g and BAP have been shown to activate microglia, which can then become functional antigen presenting cells, activate the classical complement pathway, induce opsonization of damaged neurons, and promote chemotaxis (12,37,127,131,133). However, the exact

TABLE 3B CORRELATION MATRIX FOR AD PAIRED SERA AND CSF SAMPLES

# Sera AAb Sera IgM CSF Alb Q IgG CSF IgG CSF IgM CSF IgA #CSF AAbs

# Sera AAb

Sera IgM

CSF IgG

CSF IgM

CSF IgA

# CSF AAbs

1.000000 0.456071 20.005681 0.108221 20.206542 0.003966 20.017177 0.354787

0.456071 1.000000 0.026739 0.014678 20.380856 20.050045 20.482266 0.203571

20.206542 0.058469 0.787688 0.743042 1.000000 0.129859 0.603666 0.036842

0.003966 0.457717 0.316357 0.408221 0.129859 1.000000 0.593054 0.442104

20.017177 0.207945 0.607879 0.530787 0.603666 0.593054 1.000000 20.001553

0.354787 20.026739 0.174558 0.244178 0.036842 0.442104 20.001553 1.000000

AUTOANTIBODIES IN NEURODEGENERATION

211 TABLE 4A

SUMMARY OF PD PAIRED SERA AND CSF Patient #

# CSF AAbs

# Sera AAbs

CSF IgG*

Sera IgG**

IgG Synth

IgG (loc)

CSF Alb*

Sera Alb**

Q Alb

1 2 3 4 5 6 7 8 9 10 11 % Abnormal

1.0 0.0 0.0 0.0 1.0 2.0 0.0 2.0 1.0 0.0 1.0 54.5

2.0 3.0 0.0 0.0 1.0 4.0 0.0 2.0 2.0 0.0 1.0 63.6

3.4 18.6 9.2 4.3 3.4 27.0 4.1 11.0 9.7 3.8 5.6 45.5

2.1 2.3 0.6 0.9 1.6 1.5 1.0 2.2 1.3 0.5 0.9 27.3

2.7 36.1 12.2 8.2 1.9 64.4 3.1 19.8 10.6 2.8 15.3 63.6

20.216 1.75 7.95 0.202 1.46 4.82 0.609 2.70 1.63 0.097 1.24 27.3

19.6 20.0 52.8 15.3 11.2 62.0 14.1 43.9 19.4 13.8 15.5 27.3

6.9 1.8 2.1 2.5 2.7 3.0 2.4 7.0 1.9 2.2 2.3 16.7

0.87 11.11 25.12 6.12 4.15 20.67 7.40 19.8 8.10 5.41 5.98 54.5

CSF IgM

CSF IgA*

Sera IgM*

0.16 0.19 0.12

1.88 0.34 0.67

698.7 155.2 152.9

0

66.7

404.3 532.8 203.7 148.4 159.0 37.5

* mg/dL ** g/dL

neurotoxic sequence of these molecular events requires further clarification, particularly the relative contributions of local inflammation and autoimmunity (37,55,77,95,106). AAbs to HSPG, which is also localized to AD senile plaques (79,112), were detected in the sera of AD, PD, ALS, and VD at low frequencies, suggesting that HSPG is not a primary NDD-related autoantigen, but may reflect Ag spreading in some patients or could define a distinct subset of NDD patients, possibly those with vasculitis or angiitis (36,41). The clinical features associated with BAP AAbs also remain to be elucidated. MBP AAbs showed the highest frequencies in acid-dissociated CSF from ON and MS (40260%). RR-MS CSF demonstrated higher levels of MBP AAbs than CP-MS as indicated in previous reports (129,130) where anti-MBP titers correlated with exacerbation of MS. RR-MS also had much greater CSF reactivity to tubulin than did CP-MS CSF. Lower MBP AAb frequencies were found in MS sera. Moreover, MBP AAbs were also found in VD and PD sera, but not in any AD sera specimens (Table 1). The relevance of an anti-MBP response to the etiopathogenesis of VD and PD is unclear, but may reflect general demyelination. VD patient sera showed the greatest AAb multireactivity (Table 2) with 24% of sera reacting with $4 autoantigens. This may indicate loss of immune regulation at privileged nervous system sites following stroke and a greater degree of BBB damage and Ag shedding. CIC have been previously detected in the cerebral circulation of cerebrovascular disease (CVD) patients, and sera from AD patients (52). Paired CSF and sera from VD patients immediately following stroke and at follow-up should be evaluated in future investigations for CIC, AAb, and BBB functional

parameters. In both CVD and AD brain lesions, microglial activation (elevated GFAP and HLA-DR), and increased immunoglobulin and fibrinogen deposition were previously detected (122). ALS patients also demonstrate autoreactivity to gangliosides of the myelin sheath (98,116) and to voltage-gated calcium channels (VGCC) of cholinergic neurons (111). These, in combination with tubulin AAbs, may have a high sensitivity and specificity for the detection of ALS compared to CIDP and AD (8,116), although a meta-analysis of this postulate is necessary. In addition, HSPG AAbs were the second most frequent AAb found in ALS patients. ALS has been previously shown to involve B cell immune activation with IgG3 CIC formation, as well as classical complement activation (3,7,132). Spinal cord lesions in degenerative myelopathy demonstrate deposits of both complement and immunoglobulins (12); the myelopathy patient examined here did have serum NF-H AAbs. Anti-band 3 antigen AAbs reported to be present in AD (64) were not evaluated in this study, but their detection could aid the discrimination of various NDD-related autoimmunities. The occurrence of AAbs to oxidative stress-related epitopes such as advanced glycation end products (AGE) and oxidized LDL also warrants investigation in NDDs (4). Because a large majority of NDD patients contained sera and CSF AAbs, an examination of AAb multireactivity patterns (Tables 2 and 5) may thus prove useful for subclassifying certain heterogenous NDDs and ONDs. These AAb patterns might also facilitate monitoring of disease activity and defining pathogenic antibrain autoimmune responses. The necessary predictive values

TABLE 4B CORRELATION MATRIX FOR PD PAIRED SERA AND CSF SAMPLES

# Sera AAb CSF IgG Sera IgG IgG Synth Q Alb # CSF AAbs

# Sera AAb

CSF IgG

Sera IgG

IgG Synth

Q Alb

# CSF AAbs

1.000000 0.857630 0.675300 0.813082 0.107886 0.686329

0.857630 1.000000 0.198384 0.990000 0.627380 0.518200

0.675300 0.198384 1.000000 0.181306 20.537056 0.259675

0.813082 0.990000 0.181306 1.000000 0.597074 0.504870

0.107886 0.627380 20.516516 0.597074 1.000000 0.025385

0.686329 0.518200 0.259675 0.504870 0.025385 1.000000

212

TERRYBERRY ET AL. TABLE 5

AUTOANTIGEN-SPECIFIC ANTIBODY INDICES FOR PAIRED AD AND PD CSF AND SERA Autoantigen Sample

Tubulin

NF-H

GFAP

BAP

AD1 AD2 AD3 AD4 AD5 AD6 AD7 AD8 AD9 AD10 AD11 AD12 PD1 PD2 PD3 PD4 PD5 PD6 PD7 PD8 PD9 PD10 PD11

0.122 3.37* 0 0.266 0.189 2.00* 0.034 0.037 0.936 0 0 0.067 3.29* 0 0 0 0.164 0.941 0.529 2.146* 0.056 0 0

0.226 0 0.304 0 0 0 0 0 0 0.029 0 0.178 0.457 0.289 0 0 0 0.056 0.116 1.094 0 0 0

0.365 0.74 0 0.371 0 0 0.500 0 0 0.043 0 0 0 0.598 0 0 0 0 0 0 0 0.331 0.042

0 0 0.289 0 0 0 0 0 0 0.038 0 0 0 0.341 0 0 0 0.052 0 0 0 0 0

* values $2 indicative of intrathecal AAb production.

and prognostic indices will follow clinical correlations for these AAbs in NDDs. Further studies using advanced decisional statistics (8,86,90) are in progress to substantiate the claim (44,106,107) that autoimmunity can classify a subset of AD and other NDD patients, as well as provide gains in expected clinical utilities for the use of AAb test panels as diagnostic tools. Antineuronal AAbs were previously correlated to memory dysfunction in the rodent (15,97), while CICs and IL-2 are correlated to memory impairment in AD (56,113); antineuronal AAbs are also correlated with behavioral disturbances in CNS-lupus patients (25). However, none of the VD sera for which cognitive function evaluations were available showed significant correlation of AAbs with MMSE scores by rank correlation and nonlinear regression (data not shown). Comparative evaluations of paired CSF and sera from AD and PD patients indicates that BBB dysfunction is more prevalent in PD than in AD. PD samples also demonstrated greater correlation between total number of CSF and sera AAbs, which were predominantly IgG. The degree of BBB damage in AD reported here is lower than that seen in a previous study (75), although different criteria were used. However, comparison of Q Alb values from another study (43) indicates a lower prevalence of BBB damage in the AD patients (8% vs. 25%). Adjustment for BBB leakage indicates that IgG(loc) values were elevated in 0% of AD and 27% of PD; putative intrathecal synthesis of tubulin AAbs was seen in 17% of AD and 18% of PD paired samples. The tubulin autoreactivity results suggest that tubulin AAbs, whether intrathecally produced, transudated, or leaked into the CNS, might have a pathologic or neurotoxic effect in NDDs. More detailed studies on the influence of tubulin AAbs on axonal transport (61,80), and the possible role of tubulin in NFT formation (53,84,92) are necessary. The role of the BBB in AD, and more so in PD, deserves careful

FIG. 1. Immunoblot confirmation of NF-H and Tubulin AAb reactivity and NF-H AAb phospho-epitope dependence. A) Phospho-NF-H immunoreactivity in normal (N) and AD hippocampal extracts. B) NDD and OND sera autoreactivity.

AUTOANTIBODIES IN NEURODEGENERATION

213

attention in terms of specific transport or extravasation of immunoglobulins and B cells. In MS, microglial activation by oligodendrocytes results in alterations in the phagocytic processes of BBB-forming astroglial perivascular sheets (135). GFAP1 reactive microglia could thus become the targets of CIC and antiGFAP AAbs (127). After cerebellar lesioning, GFAP1 Bergmann glia alter proteoglycan (tenascin) synthesis and axonal transport of NF (136). These alterations are also relevant to NF-H and HSPG AAb formation (133). Moreover, any one of several AAbs can stimulate macrophage-mediated oligodendrocyte injury and antibody-dependent complement-mediated cytotoxicity (135). The potential interactions of soluble APP, BAP, CIC, and AAb also needs attention. Finally, microglia-mediated complement activation, and resultant MAC formation in NDDs might, as C9 levels are in MS, be inversely correlated with IgG synthesis rates (135). Overall, the findings of this study indicate that AAbs to NF-H, tubulin, GFAP, MBP, and BAP are commonly found in some NDDs and ONDs, including motor neuron diseases. These data highlight the AAbs that should be studied for clinical utility

(8,86,90) in the subgrouping, differentiation (49), and prognosis of neurodegenerative diseases. Testing of an expanded panel of NDD phenotypes for these AAbs is warranted. Both inflammation of the CNS and antibrain autoimmunity involving BBB disturbances, complement, CICs, AAbs, T cells, and macrophages/microglia are involved in dementia, neurodegeneration, and neuropathy (1–3,5, 12,28,37,42,58,77–79,87,89,95,97,127,131). The exact molecular dissection and sequence of events leading to these immune responses, as well as their interactions are ongoing and important areas of research. ACKNOWLEDGMENTS

The authors would like to thank all specimen contributors: Drs. David Lacomis, Mark Fisher, Steve Jacobson, Toni Pallidino, A.R. Bradwell, Robert Baumhefner, Felicia Gaskin; NNRSB, as well as Drs. Shu Guang Li and Paula D’Amore, Linda Dearing, for her critical review of the manuscript, Rose Yesowitch, for her preproduction and processing of the manuscript, and the excellent library assistance of Mr. Paul Lomax.

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