A possible role for humoral immunity in the pathogenesis of - Brain

doi:10.1093/brain/awh625 Brain (2005), 128, 2665–2674

A possible role for humoral immunity inthe pathogenesis of Parkinson’s disease

Carolyn F. Orr,1 Dominic B. Rowe,2 Yoshikuni Mizuno,3 Hideo Mori3 and Glenda M. Halliday1

1Prince of Wales Medical Research Institute, University of New South Wales, Randwick,2Department of Neurology, Royal North Shore Hospital, University of Sydney, Sydney, NSW, Australia and3Department of Neurology, Juntendo University School of Medicine, Tokyo, Japan

Correspondence to: Professor G. M. Halliday, PhD, Prince of Wales Medical Research Institute,University of New South Wales, Randwick, Sydney, NSW 2031, AustraliaE-mail: [email protected]

The pathogenesis of idiopathic Parkinson’s disease is unknown, but nigral degeneration and depigmentationare associated with microglial inflammation and anti-inflammatory medications appear to protect against thedisease. The possibility that humoral immunity may play a role in initiating or regulating the inflammation hasbeen suggested by experimental studies triggering dopamine cell death using a variety of transfer strategies andthe observation of CD8+ T lymphocytes and complement in the nigra in Parkinson’s disease. We analysed theassociation between degeneration and humoral immune markers in brain tissue of patients with idiopathic (n =

13) or genetic (n = 2 with a-synuclein and n = 1 with parkin mutations) Parkinson’s disease and controls withoutneurological disease (n = 12) to determine the humoral immune involvement in Parkinson’s disease. Formalin-fixed tissue samples from the substantia nigra and primary visual cortex for comparison were stained for a-synuclein, major histocompatibility complex II (HLA), immunoglobulin M (IgM), immunoglobulin G (IgG), IgGsubclasses 1–4 and IgG receptors FcgR I–III. Antigen retrieval and both single immunoperoxidase anddouble immunofluorescence procedures were employed to determine the cell types involved and their patternand semiquantitative densities. Significant dopamine neuron loss occurred in all patients with Parkinson’sdisease, negatively correlating with disease duration (r = �0.76, P = 0.002). Although all patients had increasedinflammatory HLA immunopositive microglia, the degree of inflammation was similar throughout the disease(r = 0.08, P = 0.82). All patients with Parkinson’s disease had IgG binding on dopamine neurons but not IgMbinding. Lewy bodies were strongly immunolabelled with IgG. A mean 30 6 12% of dopamine nigral neuronswere immunoreactive for IgG in Parkinson’s disease with the proportion of IgG immunopositive neuronsnegatively correlating with the degree of cell loss in the substantia nigra (r = �0.67, P < 0.0001) and positivelycorrelating with the number of HLA immunopositive microglia (r = 0.51, P = 0.01). Most neuronal IgG was theIgG1 subclass with some IgG3 and less IgG2 also found in the damaged substantia nigra. The high affinityactivating IgG receptor, FcgRI, was expressed on nearby activated microglia. The low affinity activating IgGreceptor, FcgRIII was expressed on cells morphologically resembling lymphocytes, whereas immunoreactivityfor the inhibitory IgG receptor FcgRII was absent in all cases. This pattern of humoral immune reactivity isconsistent with an immune activation of microglia leading to the targeting of dopamine nigral neurons fordestruction in both idiopathic and genetic cases of Parkinson’s disease.

Keywords: Parkinson’s disease; microglia; humoral immunity; neuropathology

Abbreviations: IgG = immunoglobulin G; IgM = immunoglobulin M; SN = substantia nigra pars compacta

Received September 3, 2004. Revised July 30, 2005. Accepted August 1, 2005. Advance Access publication October 11, 2005

IntroductionThe pathogenesis of idiopathic Parkinson’s disease is cur-

rently unknown, but at the cellular level, significant microglial

inflammation is observed in the region of dopaminergic

degeneration (Orr et al., 2002; Hunot and Hirsch, 2003)

and some protection against its development occurs when

long-term anti-inflammatory medications are taken (Chen

et al., 2003). Microglia are the main immunocompetent

cell within the CNS (Aloisi, 2001), capable of antigen

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presentation to lymphocytes (Kreutzberg, 1996) and exchange

with blood macrophages (Flugel et al., 2001). The observation

that small numbers of CD8+ T lymphocytes occur in prox-

imity to degenerating nigral neurons (McGeer et al., 1988)

and that components of the classical or antibody-triggered

complement cascade occur in nigral Lewy bodies (Yamada

et al., 1992) in patients with Parkinson’s disease suggests

that the pathological process may involve immune-

mediated mechanisms.

Humoral immune mechanisms can trigger microglial-

mediated neuronal injury in animal models of Parkinson’s

disease (He et al., 2002), although there is currently a lack of

direct evidence that humoral immunity might be involved in

the selective death of dopamine neurons in Parkinson’s dis-

ease. We, therefore, compared the degenerating dopaminergic

substantia nigra against the unaffected non-dopaminergic

primary visual cortex in idiopathic Parkinson’s disease

and control patients for the presence of immunoglobulin

M (IgM), immunoglobulin G (IgG), IgG subclasses 1–4

and IgG receptors FcgRI–III. Although most Parkinsonian

patients have sporadic disease, mutations in a-synuclein

and parkin cause inherited forms of Parkinsonism (Huang

et al., 2004) and we also evaluated brain tissue from

patients with mutations in these genes for IgG and FcgRI

expression.

Materials and methodsPatientsAll idiopathic Parkinson’s disease patients were participants in the

Parkinson’s New South Wales brain donor programme at the Prince

of Wales Medical Research Institute [n = 13, duration 14 6 6 years;

mean age 75 6 7 years: 4 of these died early (Hoehn and Yahr stages

1–3) from intercurrent illnesses and the other 9 died at end-stage

(Hoehn and Yahr stages 4–5)]. Three familial Parkinson’s disease

cases were assessed: two had a-synuclein A53T gene mutations

(durations 1 and 9 years, Hoehn and Yahr stages 1.5 and 4, aged

47 and 53 years respectively) (Spira et al., 2001) and one had a parkin

gene mutation (duration 38 years; Hoehn and Yahr stage 5; aged

62 years) (Mizuno et al., 2001). Each clinical diagnosis of idiopathic

Parkinson’s disease was made by a movement disorder subspecialist

neurologist and required the presence of at least two of the following

cardinal signs: tremor, rigidity, bradykinesia and postural instability

as well as a positive response to levodopa (Gelb et al., 1999). For each

case, standardized neurological assessment occurred prospectively

on a yearly basis. Responsiveness to and doses of levodopa were

noted and disease severity formally staged using the Hoehn and

Yahr scale. Prospective written consent for autopsy was obtained

from all patients and their next of kin, and the project was approved

by the Human Ethics Committee of the University of New South

Wales under the Human Tissue Act of the State of New South Wales.

After death a detailed neuropathological examination was conducted

with application of current diagnostic criteria for idiopathic

Parkinson’s disease (depigmentation, cell loss and Lewy bodies in

the substantia nigra and locus coeruleus) (Gelb et al., 1999) and at

this time, prospective data were validated by retrospective question-

naires to relatives and treating physicians. All other neurological and

neurodegenerative diseases were excluded, as were cases with head

injury, brain tumour, infarction or systemic sepsis.

ControlsTwelve age-matched controls with no history of neurological or

psychiatric symptoms and no neuropathological abnormalities

were selected (aged 75 6 9 years). These controls underwent the

same clinical and neuropathological follow-up as the Parkinson’s

disease cases with the same standardized recording procedures.

The demographic details of both patients and controls are shown

in Table 1. There was no difference (unpaired t-tests, P > 0.05)

between the groups in either mean age at death or post-mortem

delay (14.5 h for cases and 19.9 h for controls). No patient or control

had haematological, immune or inflammatory disorders, or was

taking immunosuppressive medication at time of death.

Tissue preparationAfter autopsy the brains were immersion fixed in 15% buffered

formalin for 2 weeks. The brainstem was dissected from the cerebrum

at the level of the rostral midbrain, and then both cerebrum and

brainstem were embedded separately in 4% agarose and cut on a

rotary slicer into 3 mm coronal and transverse sections, respectively.

Tissue samples were taken for neuropathological diagnosis, as pre-

viously described (Halliday et al., 1996). For the present study, blocks

were taken from both the midbrain at the level of the exiting third

nerve and from the primary visual cortex and stored in 10% buffered

formalin. Idiopathic Parkinson’s disease and control midbrain and

visual cortex were cryoprotected in 30% buffered sucrose solution,

then frozen in mounting medium at �50�C, serially sectioned at

20 mm on a cryostat and mounted onto silanized slides. Idiopathic

and familial Parkinson’s disease midbrain tissue was paraffin embed-

ded, sectioned at 10 mm on a microtome and then deparaffinized.

ImmunohistochemistrySections were defatted, rehydrated in alcohols, and antigen retrieved

in 4% aluminium chloride, as previously described (Shepherd et al.,

2000). Routine immunoperoxidase staining was performed for 48 h

at 4�C with a variety of primary antibodies (Table 2), detected with

biotinylated secondary antibody (Vector, Burlingame, CA, USA) at a

1:200 dilution for 2 h at 37�C, followed by incubation in the tertiary

complex (PK-6100, ABC Vectastain Elite Kit, Vector, Burlingame,

CA, USA) at a 1:500 dilution for 2 h at room temperature. Slides were

then incubated in Vector NovaRed (SK-4800, Vector, Burlingame,

CA, USA) for 10–30 min to visualize the tertiary complex. Sections

were then washed, dehydrated through graded ethanols to xylene,

coverslipped with DePeX and allowed to dry. Human tonsillar

tissue was used as positive control for the immunological markers

analysed. Omitting primary antibodies produced appropriate

negative controls.

Double labelling fluorescent immunohistochemistry was per-

formed to determine the identity of IgG, CD64 and CD16 immuno-

positive cells. Primary antibodies (Table 2) were mixed together and

applied to the sections for 48 h at 4�C, then detected with host

specific secondary fluorescent antibodies (Table 2) mixed together

for 2 h at 37�C. Sections were coverslipped with glycerol and analysed

using a Leica DM IRB confocal laser scanning microscope and an

Olympus BX51 fluorescence microscope fitted with specific filter

systems. The cross-reactivity and specificity of the fluorescent reac-

tions were tested by incubating each primary antibody singly with the

secondary antibody solution containing two fluorophores. In these

experiments fluorescence microscopy revealed that only the appro-

priate fluorophore labelled the primary antibody with no cross-

reactivity with the second fluorophore observed.

2666 Brain (2005), 128, 2665–2674 C. F. Orr et al.

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Table 1 Case details

Case Gender(M/F)

Stage(0–5)

Onset age(years)

Age at death(years)

Disease duration(years)

Cause of death Post-mortemdelay (h)

asyn1 M 1.5 46 47 1 Asphyxiation <24asyn2 M 4 44 53 9 Pneumonia 18parkin M 5 24 62 38 Cardiorespiratory death <24PD1 M 2 44 55 11 Colon cancer 6PD2 F 2 68 78 10 Pancreatic cancer 17PD3 M 2 82 85 3 Prostatic cancer 23PD4 M 3 66 70 4 Cardiorespiratory death 15PD5 M 4 55 72 27 Myocardial infarction 30PD6 M 4 58 78 20 Pneumonia 3PD7 F 4 67 77 10 Pneumonia 8PD8 M 4 72 84 12 Pneumonia 14PD9 F 5 49 76 27 Cardiorespiratory death 18PD10 M 5 53 69 16 Myocardial infarction 15PD11 M 5 57 72 15 Perforated gastric ulcer 12PD12 M 5 66 76 10 Pneumonia 4PD13 M 5 66 81 15 Pneumonia 24C1 F 0 – 57 – Ovarian cancer 19C2 M 0 – 61 – Myocardial infarction 14C3 F 0 – 69 – Ruptured aortic aneurysm 4C4 F 0 – 71 – Perforated gastric ulcer 14C5 M 0 – 72 – Pneumonia 5C6 F 0 – 75 – Colon cancer 29C7 F 0 – 77 – Myocardial infarction 26C8 F 0 – 82 – Bronchiectasis 29C9 M 0 – 82 – Malignant melanoma 45C10 M 0 – 83 – Colon cancer 23C11 F 0 – 86 – Myocardial infarction 7C12 F 0 – 90 – Colon cancer 24

Table 2 Details of antibodies used in immunohistochemistry

Antigen Catalogue #, dilution Species

Primary antibodiesa-synucleina,b 610787c, 1:200 Mouse anti-humanHLA-DP/DQ/DR (major histocompatibility class II)a M0775d, 1:500/100 Mouse anti-humanIgMa MCA1162, 1:500 Mouse anti-humanIgGa,b,e,f AHP526, 1:40 Rabbit anti-humanIgG class 1a MCA514G, 1:250 Mouse anti-humanIgG class 2a MCA515G, 1:250 Mouse anti-humanIgG class 3a MCA516G, 1:250 Mouse anti-humanIgG class 4a MCA2098G, 1:50 Mouse anti-humanCD64 (high affinity activating IgG receptor FcgRI)a,g 216-020h, 1:75/200 Mouse anti-humanCD16 (low affinity activating IgG receptor FcgRIII)a,e,f MCA617, 1:100 Rat anti-humanCD32 (inhibitory IgG receptor FcgRII)a MCA1075, 1:1000 Mouse anti-humanMSR (macrophage scavenger receptor 1)g AB5486i, 1:5000 Goat anti-humanCNPase (20, 30-cyclic nucleotide 30-phosphodiesterase)f C5922j, 1:1500 Mouse anti-humanp25a (Lindersson et al., 2005)f giftk, 1:200 Rabbit anti-human

Secondary fluorescence antibodiesFluoresceinb N1034l, 1:50 Donkey anti-rabbitFluoresceing 705-095-147m, 1:200 Donkey anti-goatAlexa Fluor 594b,g A-21203n, 1:250 Donkey anti-mouseAlexa Fluor 568e,f A-11011n, 1:250 Goat anti-rabbitAlexa Fluor 488f A-11001n, 1:500 Goat anti-mouseAlexa Fluor 488e,f A-21208n, 1:500 Donkey anti-rat

Immunoperoxidase experimentsa, double-labelling immunofluorescence experiments for Lewyb or immunee pathology, for microgliag or foroligodendrogliaf. Antibodies from Serotec Ltd, Oxford, UK unless indicated; cTransduction Laboratories, Lexington, USA, dDako, Glostrup,Denmark, hAncell, Bayport, USA, iChemicon, Temecula, USA, jSigma, St Louis, USA, kProfessor Poul Jensen, Department of MedicalBiochemistry, University of Aarhus, Denmark, lAmersham Pharmacia Biotech, Buckinghamshire, UK, mJackson ImmunoresearchLaboratories, West Grove, USA, nMolecular Probes, Eugene, USA.

IgG targeting of neurons in Parkinson’s disease Brain (2005), 128, 2665–2674 2667

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AnalysisThe degree of pigmented cell loss in the substantia nigra pars com-

pacta (SN) was evaluated using a previously published areal fraction

method (Halliday et al., 1996). Briefly, the area occupied by pig-

mented neurons in the SN was measured and the areal fraction

occupied by pigmented neurons determined for each case by

point counting using an 11 · 11 eyepiece grid on ·400 magnification.

The area was multiplied by the fraction to determine the fraction of

the SN occupied by pigmented cells, and the data expressed as a

percentage of mean control values. Repeated measurements by the

same or different investigators gave an average variance in the area of

the SN of 3–7%. The degree of microglial activation was evaluated

using a previously published areal fraction method (Shepherd et al.,

2000). The areal fraction occupied by HLA-DP/DQ/DR-immunore-

active microglia in two SN sample regions of maximum staining

intensity was determined for each case by point counting using

an 11 · 11 eyepiece grid on ·200 magnification. An average variance

of 5–5.5% was demonstrated in repeated measures by the same or

different investigators. Areal fraction measurements were used for

quantitation as these describe the visual representation of cell

densities.

The proportion of IgG-immunopositive to total pigmented SN

neurons was quantified in each case and control at ·200 magnifica-

tion. An average variance of 3–5% was demonstrated in repeated

measures by the same or different investigators. The degree of cellular

immunoreactivity in the pigmented region of the SN and two

randomly chosen areas of the visual cortex was evaluated using

semiquantitative visual grading (none, mild, moderate, severe) at

·100 magnification (with ·200 magnification used for confirmation)

consistent with standard neuropathological evaluations. There were

no differences in the grade given by the same or different investi-

gators on different days.

Statistical differences between groups were evaluated using

Mann–Whitney U-tests (StatView software) and correlations

between variables evaluated using Spearman rank tests. A P-value

of <0.05 was accepted as the level of significance.

Fig. 1 Cell loss (A and B), Lewy body formation and microglia activation (D and E) in the dopaminergic SN in Parkinson’s disease (B–E)compared with a control (A). (A–C) Sections of the SN immunohistochemically stained with antibodies to a-synuclein (a-syn) showingneuromelanin pigmented neurons. Scale in B is equivalent for A. There is an obvious loss of pigmented dopamine neurons from theventrolateral SN of patients with Stage 2 Parkinson’s disease (B) compared with controls (A). Some remaining pigmented dopamineneurons in the SN of patients with idiopathic Parkinson’s disease contain a-synuclein-immunoreactive Lewy bodies (arrowheads in C).(D and E) Sections of the SN immunohistochemically stained with antibodies to HLA-DP/DQ/DR (HLA), a marker for the majorhistocompatibility complex class II protein. Scale in E is equivalent for D. HLA-immunoreactive upregulated microglia (arrows) nearnon-immunoreactive pigmented SN neurons (asterisks) in thick (D) and thin (E) midbrain sections from cases with idiopathic Parkinson’sdisease (D and E) and from Parkinson’s disease cases with a-synuclein (inset in D) and parkin (inset in E) gene mutations.

2668 Brain (2005), 128, 2665–2674 C. F. Orr et al.

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ResultsAs expected, both idiopathic and genetic Parkinson’s disease

cases had significant pigmented cell loss (average 83% SN

cell loss, P < 0.0001) and a significant increase in HLA-

immunopositive microglia (average 37% of SN area occupied

by activated microglia, P = 0.04) in the SN compared with

controls (Fig. 1). The increase in HLA-immunoreactive

microglia in Parkinson’s disease SN was considered specific

owing to the lack of upregulation observed in control SN

(Table 3) and control and Parkinson’s disease visual cortex

(data not shown). Idiopathic and a-synuclein gene mutation

cases had Lewy body formation in some of the remaining

pigmented SN neurons (Fig. 1C), whereas no Lewy body

formation was seen in the few remaining SN neurons in

the parkin gene mutation case. Parkinson’s disease cases at

earlier disease stages had less cell loss than those with end stage

disease (Table 3, P = 0.005) with greater SN cell loss corre-

lating with increasing disease duration (R = 0.76, P = 0.002).

In contrast, there was no significant difference between the

number of HLA-immunopositive microglia over the disease

course (Table 3, P = 0.39) or with longer disease durations

(r = 0.08, P = 0.82) consistent with a steady inflammatory

response throughout the disease.

Immunopositive staining was observed on some pigmented

dopamine neurons in the SN using antibody to IgG but not

to IgM. Double label immunofluorescence experiments

showed that IgG co-localized with a-synuclein in pigmented

SN neurons (Fig. 2A–F). Confocal microscopy revealed IgG

concentrating at the cell surface membrane of Parkinson’s

disease dopamine neurons (Fig. 2A and B) and also on

their Lewy bodies (Fig. 2C–F). IgG-immunopositive pig-

mented neurons and neurites were found in the SN of all

idiopathic patients and in the a-synuclein and parkin gene

mutation patients, but not in control SN or in idiopathic

Parkinson’s disease visual cortex (Fig. 2G–K). A mean 30 6

12% of idiopathic Parkinson’s disease SN neurons were

immunoreactive for IgG with significantly more IgG

immunopositive neurons in early-stage compared with

end-stage disease (Table 3, P = 0.003). The proportion of

IgG-immunopositive neurons negatively correlated with

the degree of SN cell loss (R = �0.67, P < 0.0001) and posi-

tively correlated with the number of HLA-immunopositive

microglia (r = 0.51, P = 0.01). On average, �4% of remaining

pigmented neurons contained Lewy bodies in idiopathic

cases and double labelling revealed that all IgG-immuno-

positive pigmented neurons containing Lewy bodies had

both proteins (a-synuclein and IgG) in the inclusions

(Fig. 2C–F). Analysis of the IgG subclass specificity of this

response showed significant immunoreactivity for IgG1 on a

large proportion of degenerating SN neurons in most cases

(Table 3). There was less immunoreactivity for IgG3 and even

less IgG2 on neurons (Table 3). Immunoreactivity for IgG4

was absent in all cases.

The high affinity receptor FcgRI (CD64) was present on

large amoeboid-shaped cells near the pigmented neurons in

a-synuclein and parkin gene mutation Parkinson’s disease

and in 11/12 idiopathic patients, but not in control SN or

in Parkinson’s disease visual cortex (Fig. 3A–E). Double label

immunofluorescence proved the identity of the FcgRI-

immunopositive amoeboid-shaped cells as activated phago-

cytic microglia bearing pigment remnants (Fig. 3F–I). No

immunoreactivity for the inhibitory IgG receptor FcgRII

(CD32) was found in any midbrain or cortical section tested

in any case. The low affinity IgG receptor FcgRIII (CD16) was

present on small SN cells in all cases (Fig. 4A) and in 7/12

controls (Fig. 4B). Double labelling experiments revealed that

these small cells were not oligodendroglia but morpho-

logically resembled lymphocytes (Fig. 4F and G). No

oligodendroglia or FcgRIII-immunopositive cells were IgG-

immunopositive (Fig. 4D, E, H and I). No FcgRIII

immunoreactivity was found in the other controls or in the

visual cortex (Fig. 4C).

Table 3 Mann–Whitney U analysis of variables by group

Controls Stages 1–3 Stages 4 and 5 P-value

n 12 5 11 �Mean age (years 6 SD) 75 6 9 67 6 16 74 6 9 >0.68Mean age of onset (years 6 SD) � 61 6 16 56 6 13 0.50Disease duration (years 6 SD) � 5.8 6 4.4* 18.1 6 9.1* 0.02Mean % SN cell loss (6 SD) 0 6 20 75 6 3*,# 87 6 4*,# <0.004Mean % HLA + microglia in SN (6 SD) 7 6 12 53 6 18# 30 6 33 0.002Density of IgM + SN cells 0 0 0 �Mean % IgG + SN cells (6 SD) 0 6 0 44 6 9*,# 24 6 6*,# <0.004Density of IgG1 + SN cells 0 + to +++ 0 to +++ �Density of IgG2 + SN cells 0 0 to +++ 0 to +++ �Density of IgG3 + SN cells 0 0 to +++ 0 to ++ �Density of IgG4 + SN cells 0 0 0 �Density of FcgRI + SN cells 0 0 to ++ + to +++ �Density of FcgRII + SN cells 0 0 0 �Density of FcgRIII + SN cells 0 to + + to +++ + to +++ �

*Different from other Parkinson’s disease group. #Different from controls. + = A few positive cells; ++ = moderate numbers ofpositive cells; +++ = large numbers of positive cells.


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DiscussionThe idiopathic cases analysed in the present study had typical

levodopa responsive Parkinson’s disease. The degree of cell

loss and reactive microgliosis were similar to those previously

described for similar case types (Fearnley and Lees, 1991;

Imamura et al., 2003). Although cell loss increased with

disease duration, microglial upregulation remained relatively

constant throughout the disease course. Similar in vivo find-

ings show that the specific, early upregulation of SN microglia

in Parkinson’s disease correlates with disease severity and

dopamine terminal loss, but not with disease duration

(Ouchi et al., 2005). The activation of microglia in the SN

in Parkinson’s disease is not a generalized inflammatory

response in the brain, but is highly localized (see also

Ouchi et al., 2005), contrasting with the overall small increase

in the immune response of microglia to ageing (Overmyer

et al., 1999). These findings suggest that an inflammatory

reaction may contribute to the pathogenesis of this disease.

Fig. 2 IgG binding to SN dopamine neurons in Parkinson’s disease. Asterisks mark non-immunoreactive neuromelanin pigment, a markerfor human dopamine neurons. Arrowheads show Lewy bodies. (A–F) Double labelling immunofluorescence for a-synuclein (a-syndetected with Alexa Fluor 594) and IgG (detected with fluorescein) in pigmented SN neurons. Scale in F is equivalent for A–F. Confocalimages show IgG (A) concentrating at the cell surface membrane of an a-synuclein (B) positive dopamine neuron in a patient withidiopathic Parkinson’s disease. Fluorescence and brightfield microscopy shows IgG (C) colocalizing with a-synuclein (D) in Lewy bodies ina non-fluorescent (E) pigmented (F) dopamine neuron in the SN of a patient with idiopathic disease. (G–K) Sections of the SNimmunohistochemically stained with antibodies to IgG (detected with Vector NovaRed) showing immunopositive and immunonegativeneuromelanin pigmented neurons. Scale in K is equivalent for G–K. IgG positive (red) and negative (asterisks) pigmented dopamine neuronsand neurites were found in the SN of a-synuclein (G), parkin (H) and idiopathic (I) Parkinson’s disease patients. No IgG immunoreactivitywas observed on pigmented neurons (asterisks) in control midbrain (J) or on neurons in Parkinson’s disease visual cortex (K).

2670 Brain (2005), 128, 2665–2674 C. F. Orr et al.

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In both idiopathic and genetic cases of Parkinson’s disease,

we found pigmented dopamine neurons immunolabelled

with IgG and associated with an increase in activated

microglia expressing the high affinity IgG receptor FcgRI.

In the same tissue we found FcgRI-immunopositive SN

microglia containing pigment granules consistent with a

phagocytic attack on the IgG-immunopositive pigmented

neurons. These immunological changes peaked at early

disease stages when the disease mechanism is thought to be

most active (Clarke et al., 2000). These significant immune

changes in Parkinson’s disease might be merely associated

with the disease as epiphenomena, or alternatively could be

involved in disease pathogenesis.

There is some evidence for the concept that IgG binding

might be a generic response to the death of CNS neurons.

This process may increase with age owing to the known hum-

oral changes occurring over time (Ginaldi et al., 1999a–c ;

Boren and Gershwin, 2004). These changes include the expan-

sion of natural killer cells and of T cells which progressively

acquire phenotypes intermediate between T lymphocytes and

natural killer cells, decreases in circulating memory B cells,

and T-cell dysfunction associated with reduced thymic gen-

eration of naive T cells, virus-induced expansion of terminal

effectors and increased levels of memory cells producing type I

and II cytokines. In addition, circulating autoantibodies are

increased with age (Boren and Gershwin, 2004) and do bind

non-specifically to degenerating neurons under conditions of

traumatic (Stein et al., 2002) or degenerative (D’Andrea,

2005) blood–brain barrier compromise. However, the IgG

binding following trauma or in Alzheimer’s disease is only

found on neurons in the advanced stages of degeneration and

only in the vicinity of the blood–brain barrier compromise. In

our Parkinson’s disease cases, IgG coated both damaged

(containing Lewy bodies) and apparently undamaged neu-

rons, with some neurons having extensive immunolabelling of

their dendritic tree (Fig. 2). None of the cases examined had

evidence of blood–brain barrier compromise, consistent with

specific studies on this issue (Haussermann et al., 2001),

although dysfunction of midbrain efflux pumps for small

molecules has recently been identified in the blood–brain

barrier of Parkinson’s disease patients (Kortekaas et al.,

2005). Additionally, the degree of IgG labelling did not

Fig. 3 Reactive microglia (arrowed) around SN dopamine neurons in Parkinson’s disease. Asterisks mark non-immunoreactiveneuromelanin pigment, a marker for human dopamine neurons. (A–E) Sections of the SN immunohistochemically stained with antibodiesto the high affinity activating IgG receptor FcgRI (CD64 detected with Vector NovaRed) showing immunopositive microglia nearimmunonegative neuromelanin pigmented neurons. Scale in B is equivalent for A. Scale in E is equivalent for C–E. FcgRI-immunoreactivemicroglia were found in the SN of parkin (A), a-synuclein (B), and idiopathic (C) Parkinson’s disease patients, but were absent incontrol nigra (D) and in Parkinson’s disease visual cortex (E). (F–I) Double labelling immunofluorescence for macrophage scavengerreceptor type 1 (MSR detected with fluorescein) and FcgRI (CD64 detected with Alexa Fluor 594) in SN microglia of a patient withidiopathic disease. Scale in I is equivalent for F–I. Fluorescence and brightfield microscopy shows macrophage scavenger receptor(F) co-localized with FcgRI (G) on a nonfluorescent (H) glial cell containing pigment remnants (I), confirming the FcgRI cells asactivated phagocytic microglia.


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increase over time, as may be expected if there is a continual

blood–brain barrier problem, or decrease suddenly, as may be

expected with a transient breakdown then recovery of the

barrier. On the contrary, the neuronal IgG labelling related

to the degree of neuronal loss and microglial activation. In the

absence of a compromised blood–brain barrier to large mole-

cules, these findings suggest that IgG coating is a part of the

disease phenotype occurring prior to the gross degeneration

of the pigmented SN neurons.

The second possibility is that humoral immune mecha-

nisms play a role in initiating the selective removal of dopa-

mine SN neurons in Parkinson’s disease. Antibody coating

(opsonization) of a cell can enable its ingestion and degrada-

tion through interaction with the Fc receptors of phagocytic

macrophages, and can also activate the complement system

via the classical complement cascade (Roitt et al., 2001).

Rather than simply a disease epiphenomenon, neuronal

IgG may be involved in the pathogenesis of dopamine

neuronal death by triggering either complement activation

or attack by surrounding microglia. There is experimental

evidence that humoral mechanisms can mediate damage to

the SN through both mechanisms. Previous studies have

demonstrated components of the classical (antibody-

triggered) but not the alternative complement cascade on

Lewy bodies in Parkinson’s disease (Yamada et al., 1992).

The potential relevance of complement activation was

shown by Defazio et al. (1994), who demonstrated that adding

serum from Parkinson’s disease patients to mesencephalic

dopamine neurons in culture produced a reduction in dopa-

mine neuronal function and viability only in the presence of

complement. Though the identity of the substance in serum

causing complement activation was not identified, our data

suggest it could be IgG. Similar in situ targeting of selective

neurons by IgG and patient serum occurs in the immune-

mediated disease human T-lymphotropic virus Type 1 asso-

ciated myelopathy/tropical spastic paraparesis and correlates

with decreased neuronal firing, neuronal damage and disease

progression (Jernigan et al., 2003; Kalume et al., 2004; Levin

et al., 1998, 2002). In the present study, there was a strong

association between neuronal IgG labelling in Parkinson’s

disease and the progression of neurodegeneration.

In vivo immunization of guinea pigs with bovine mesen-

cephalic homogenates (Appel et al., 1992) or hybrid dopa-

mine cell line homogenates (Le et al., 1995) causes selective

Fig. 4 The low affinity IgG receptor FcgRIII (A–D, F) in the SN (A, B, D–I) and visual cortex (C). Asterisks mark non-immunoreactiveneuromelanin pigment, a marker for human dopamine neurons. (A–C) Sections of the SN immunohistochemically stained withantibodies to the low affinity activating IgG receptor FcgRIII (CD16 detected with Vector NovaRed) showing immunopositive cells (arrows)near immunonegative neuromelanin pigmented neurons (asterisks). Scale in C is equivalent for A–C. FcgRIII-immunoreactive cellsmorphologically resembling lymphocytes were found in the SN of idiopathic Parkinson’s disease (A) patients and some controls(B) but were absent in Parkinson’s disease visual cortex (C). (D and E) Double labelling immunofluorescence for FcgRIII (CD16 detectedwith Alexa Fluor 488) and IgG (detected with Alexa Fluor 568) showing IgG-immunopositive (E) but FcgRIII-immunonegative(D) SN pigmented neurons in a patient with idiopathic disease. Note the double-labelled cells in a nearby blood vessel. Scale in E isequivalent for D. (F and G) Double labelling immunofluorescence for FcgRIII (CD16 detected with Alexa Fluor 488) and p25a, anoligodendroglia marker (detected with Alexa Fluor 568) in the SN of a patient with idiopathic disease. Scale in G is equivalent for F.The FcgRIII-immunoreactive cells (arrows in F) were not oligodendroglia near pigmented neurons (arrowheads in G). (H and I) Doublelabelling immunofluorescence for CNPase, an oligodendroglia marker (detected with Alexa Fluor 488) and IgG (detected withAlexa Fluor 568) in the SN of a patient with idiopathic disease showing the specificity of IgG binding to pigmented SN neurons (I)and not oligodendroglia or their processes (H). Scale in I is equivalent for H.

2672 Brain (2005), 128, 2665–2674 C. F. Orr et al.

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damage to the SN involving microglial activation and loss

of dopamine cells. Stereotaxic injection of IgG isolated

from Parkinson’s disease patients into rodent SN induces

microglial activation followed by injury to dopamine neurons

(He et al., 2002). In these immune-mediated animal models of

the disease, IgG is observed on �30% of the dopamine neu-

rons prior to microglial activation and neuronal death (He

et al., 2002), a similar proportion to that observed in our

study. Knockout of FcgR protects mice from both microglial

activation and dopamine cell death (He et al., 2002), proving

that the interaction of neuronal IgG with its microglial recep-

tor is critical for the dopamine cell death. In Parkinson’s

disease, microglia are activated in the vicinity of degenerating

SN neurons (McGeer et al., 1988; Banati et al., 1998; Mirza

et al., 2000) and this microglial activation is a constant feature

of multiple different toxin-induced animal models of Parkin-

son’s disease (Kurkowska-Jastrzebska et al., 1999; Kim et al.,

2000; Gao et al., 2002a, b). Direct inhibition of microglial

activation (He et al., 2001; Liu et al., 2000; Wu et al.,

2002) or therapeutic immunization using adoptive transfer

of immune cells (Benner et al., 2004; Boska et al., 2005) is

neuroprotective in these models. The cross-linking of the

FcgR on microglia with antibody ligand on foreign cells

can initiate phagocytosis, antibody-dependent cell-mediated

cytotoxicity, and the release of proinflammatory cytokines

and oxygen free radicals (Ulvestad et al., 1994). Our finding

that activated microglia express high affinity activating IgG

receptors (FcgRI) in both idiopathic and genetic forms of

Parkinson’s disease suggests that the activation of microglia

may be induced by the neuronal IgG. The FcgRI-

immunopositive SN microglia contained pigment granules,

supporting their potential involvement in a phagocytic attack

on IgG immunopositive pigmented neurons. IgG binding to

dopamine neurons may result in their selective targeting and

subsequent destruction by activated microglia in idiopathic

and familial Parkinson’s disease.

Different immunoglobulin classes and subclasses mediate

specialized effector functions (Roitt et al., 2001), including

complement fixation (IgM > IgG3 > IgG1), opsonization

(IgG1 > IgG3) and antibody-dependent cellular cytotoxicity

(IgG1 > IgG3). IgM (present study) and IgE (Hunot and

Hirsch, 2003) do not label dopamine or other cell types in

Parkinson’s disease brain. The lack of IgM suggests that com-

plement fixation is not an early or dominant event. Of the IgG

subclasses, we found predominantly IgG1 followed by IgG3

deposition on pigmented SN neurons and Lewy bodies. IgG1

has the highest affinity for FcgRI with the dominant function

of this receptor being antibody-dependent cellular cytotoxi-

city (Roitt et al., 2001). The dominance of IgG1 and FcgRI

in Parkinson’s disease SN is more consistent with a role for

neuronal IgG in the direct targeting of neurons by the

surrounding microglia rather than through complement

activation. However, in myasthenia the complement pathway

is activated by IgG1 (Hughes et al., 2004) and complement

can trigger microglia through the CR3 receptor. The presence

of both antibody and complement on Parkinson’s

disease neurons may synergistically enhance the microglial

toxicity.

Death of dopamine neurons is believed to involve defective

proteolytic processing (parkin) and abnormal protein accu-

mulation (a-synuclein) in these forms of familial Parkinson’s

disease. Although the identity of the antigen or antigens

responsible for IgG binding to dopamine neurons remains

unclear, the fact that antibody-mediated cytotoxicity is pas-

sively transferable (Benner et al., 2004; Boska et al., 2005) and

acts on microglia via their Fcg receptor argues that our finding

of the presence of IgG on dopamine neurons in both typical

idiopathic and genetic forms of Parkinson’s disease is relevant

to disease pathogenesis. It is possible that the final common

pathway of these aetiologically diverse cellular injuries

involves altered expression of proteins or other macro-

molecules on the dopamine neuron surface triggering

immune recognition and IgG binding. Subsequent antigen-

induced cross-linking of FcgR on microglia triggering their

activation may underlie the mechanism behind propagation

of inflammation. Diverse insults to SN dopamine neurons

could thus set in motion a self-sustaining cascade of events

whereby dopamine neurons become the target of a selective

humoral immune-mediated injury effected through activated

microglia. We believe that antibody-directed microglial

activation may represent a common and potentially treatable

mechanism for the ultimate degeneration of dopamine neu-

rons in Parkinson’s disease.

AcknowledgementsWe thank the tissue donors who made this work possible,

David Veivers for the human tonsillar tissue, Heather

McCann for her guidance in the laboratory, Anita Ophof

for assistance with the quantitation and Heidi Cartwright

for the preparation of the figures. This study was supported

by research funds from the Parkinson’s Disease Foundation

and the National Parkinson Foundation of the United States

to G.M.H., the National Health and Medical Research

Council of Australia to G.M.H. and D.B.R., and an Australian

Government International Postgraduate Research

Scholarship to C.F.O.

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