Mechanisms of Disease: aquaporin-4 antibodies in neuromyelitis optica

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SUMMARY

Mechanisms of Disease: aquaporin-4 antibodies in neuromyelitis opticaSven Jarius, Friedemann Paul, Diego Franciotta, Patrick Waters, Frauke Zipp, Reinhard Hohlfeld, Angela Vincent* and Brigitte Wildemann

Vanderbilt Continuing Medical Education online

This article offers the opportunity to earn one Category 1

credit toward the AMA Physician’s Recognition Award.

Competing interestsA Vincent declared associations with the following companies: Athena Diagnostics and RSR Ltd. See the article online for full details of the relationships. The other authors declared no competing interests.

INTRODUCTIONNeuromyelitis optica (NMO or Devic syndrome) was first described in the late 19th century by Eugène Devic and others.1–4 In Japan and other Asian countries, NMO is often called optico-spinal multiple sclerosis (OSMS), and is more prevalent than typical multiple sclerosis (MS). Although NMO has been classified as a subtype of MS for many years, the disease is classically restricted to the optic nerves and spinal cord, and it is now clear that it has distinct clinical and pathological features. In particular, new histo pathological and sero logical findings strongly suggest the involvement of the humoral immune system, and the detection of NMO-specific serum autoantibodies, collectively known as NMO-IgG, helps to distinguish NMO from MS.5,6 The discovery of NMO-IgG, and the subsequent identification of aquaporin-4 (AQP4)—the most abundant water channel in the CNS—as its target antigen,6–9 makes NMO the first inflammatory demyelinating disorder of the CNS to have a defined autoantigen, which enables diagnosis of the disease by use of a serological test. Moreover, the possibility that NMO is an auto antibody-mediated disease, analogous to myasthenia gravis and other auto-immune channelopathies, raises the likeli hood of establishing therapeutic strategies aimed at the humoral arm of the immune system.

In this article, we will first review the clinical, sero-logical and pathological characteristics of NMO. We will then discuss the evidence for an antibody-mediated mechanism in the patho genesis of this disease.

Neuromyelitis optica (NMO) is a rare CNS inflammatory disorder that predominantly affects the optic nerves and spinal cord. Recent serological findings strongly suggest that NMO is a distinct disease rather than a subtype of multiple sclerosis. In NMO, serum antibodies, collectively known as NMO-IgG, characteristically bind to cerebral microvessels, pia mater and Virchow–Robin spaces. The main target antigen for this immunoreactivity has been identified as aquaporin-4 (AQP4). The antibodies are highly specific for NMO, and they are also found in patients with longitudinally extensive transverse myelitis without optic neuritis, which is thought to be a precursor to NMO in some cases. An antibody-mediated pathogenesis for NMO is supported by several observations, including the characteristics of the AQP4 antibodies, the distinct NMO pathology—which includes IgG and complement deposition and loss of AQP4 from spinal cord lesions—and emerging evidence of the beneficial effects of B-cell depletion and plasma exchange. Many aspects of the pathogenesis, however, remain unclear.KEYWORDS aquaporin-4 antibodies, Devic syndrome, multiple sclerosis, neuromyelitis optica, pathogenesis

S Jarius is a Neurologist and Neuroimmunologist and P Waters is a Postdoctoral Scientist in the Neurosciences Group, Weatherall Institute of Molecular Medicine, University of Oxford, UK. F Paul is a Neurologist and Senior Physician, and F Zipp is Professor of Neurology at the Cecilie Vogt Clinic for Neurology, Charité–Universitaetsmedizin Berlin, Germany. D Franciotta is Head of the Laboratory of Neuroimmunology at the Neurological Institute “C. Mondino”, Pavia, Italy. R Hohlfeld is Professor of Neurology and Clinical Neurosciences at the Ludwig Maximilian University, Munich, Germany. A Vincent is Professor of Neuroimmunology and Head of the Neurosciences Group, University of Oxford. B Wildemann is Professor of Neurology and Head of the Division of Molecular Neuroimmunology, Department of Neurology, University of Heidelberg, Germany.

Correspondence*Neurosciences Group, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford OX3 9DS, UK [email protected]

Received 27 August 2007 Accepted 9 January 2008 Published online 11 March 2008

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REVIEW CRITERIAReferences for this review were identified by searches of PubMed for articles published from 1966 to March 2007, with the terms “neuromyelitis”, “(Devic’s OR Devic) AND (syndrome OR disease)”, “opticospinal OR optico-spinal”, “multiple sclerosis AND Japan”, “longitudinal extensive transverse myelitis”, “myelitis AND optic neuritis” and “aquaporin-4”. Articles were also identified through searches of the authors’ own files. With a few exceptions (publications from the 19th century), only papers published in English were reviewed.

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CLINICAL FEATURES OF NEUROMYELITIS OPTICANMO predominantly affects the optic nerves and spinal cord.1,10,11 Brain lesions can occur during the course of the disease, but they mostly remain clinically silent.12 NMO usually begins with either myelitis or optic neuritis—Devic’s classical syndrome of simultaneous bilateral optic neuritis and myelitis occurs in only 10% of cases.11 Spinal cord symptoms in NMO range from mild sensory disturbances to complete transverse myelitis with tetraplegia or paraplegia, sensory impair-ments and bladder–bowel dysfunction. NMO usually follows a relapsing course without marked progression of disability between relapses, but in a minority of cases the disease course can be monophasic (15–23%)10,13 or secondary chronic progressive (2%).14 Spontaneous remission of neurological dysfunction is rare in NMO in comparison with MS, and accumula-tion of irreversible deficits and rapid progression of disability are frequent. Studies on the natural course of NMO report progression to severe motor dysfunction (muscle strength ≤2 on the Medical Research Council scale) or substantial loss of visual function (<20/200) in one or both eyes, in 45% of cases. Respiratory failure caused by ascending cervical myelitis is the most frequent cause of death in patients with NMO.11 Attacks tend to be particularly severe in patients with monophasic NMO, who often show simultaneous optic neuritis and myelitis. The 5-year survival rate was found to be 90% in this group.11,15 Patients with relapsing NMO were reported to have a 5-year survival rate of 70%.11

The median age at NMO onset is about 37 years,10,13,16,17 although the disease can also occur during infancy18 or in the elderly.19 There is no sex bias among patients with a monophasic disease course,11 but there is a marked female preponderance among patients with relapsing disease.

ImagingIn patients with NMO, T2-weighted MRI of the spinal cord (Figure 1A) usually shows wide-spread lesions extending over three or more vertebral segments. In acute stages of the disease, the spinal cord lesions might be accompanied by gadolinium enhancement (detectable days to months following relapse), swelling of the spinal cord, and necrosis.20 Vertical extension of spinal cord lesions over three or more segments is the most important MRI marker of NMO.10

Owing to confluent spinal lesions, however, longitudinal T2 hyperintense areas might also occur in patients with MS in rare cases.6,10,21,22 Conversely, short lesions might be found in patients with NMO when MRI is performed very early during a relapse or when the lesion is depicted in its atrophic residual stage.10 Diffusion tensor imaging shows increases in mean diffu-sivity and fractional anisotropy in spinal NMO lesions compared with MS lesions; these changes correlate with the extent of tissue damage, as well as with clinical disability.23,24 Optical co herence tomography often detects axonal injury, indi-cated by a thinning of the retinal nerve fiber layer, in patients with NMO, but it does not discriminate well between NMO and MS.25 By contrast, thicken ed vessel walls and narrow arterioles extending far into the periphery, which can be revealed by retinal photography or simple fundoscopy in patients with NMO, are largely absent in MS.25

Classically, brain MRI is normal at onset of NMO, but brain or brainstem lesions do not preclude a diagnosis of NMO. In a recent study, nonspecific, non-MS-like lesions were found in 30 out of 60 patients with an NMO disease course of several years, whereas MS-like lesions—most of them clinically silent—were present in only 10% of cases.12 Signal abnormalities in fluid-attenuated inversion recovery images in NMO tend to be less than 3 mm in diameter and do not show the perpendicular orientation typical of MS.26 Interestingly, T1 brain lesions (thought to represent persistent axonal loss in MS) are mostly missing in NMO.26 In a minority of cases, periventricular, and hypothalamic–diencephalic lesions are detectable.27

Cerebrospinal fluid analysisCerebrospinal fluid (CSF) analysis in patients with NMO typically shows mixed lymphocytic and neutrophilic pleocytosis, and eosinophils might be present in rare cases.11 The total CSF cell count can exceed 50/µl, especially when measured during relapses,11,13,15,16,28 but, in contrast to MS, the cell count might be normal or near normal during remissions. Oligoclonal bands are detectable in only about 35% of cases, and they often disappear during the course of the disease.11,15,29,30 The polyspecific, intrathecal B-cell activation (the measles–rubella–zoster reac-tion) seen in most patients with MS31,32 is absent in NMO.33 Protein 14-3-3, a marker of neuronal destruction, is elevated in the CSF of some

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patients with NMO, reflecting the vast extent of spinal tissue damage in these individuals.34

DIAGNOSIS OF NEUROMYELITIS OPTICADiagnostic criteria Until recently, the diagnosis of NMO was established according to the criteria proposed by Wingerchuk and colleagues in 1999.11 On the basis of the analysis of clinical, MRI and labora tory data from 96 patients, and the recent discover ies of NMO-IgG6,9 and AQP4 antibodies (AQP4-Abs)7,8 in patients with NMO, the

diagnostic criteria have now been revised (Box 1).10 The new criteria achieve a higher specificity regarding the differentiation between NMO and MS, and an improved sensitivity, resulting from the inclusion of NMO-IgG serology and the dismissal of non-opticospinal symptoms as an exclusion criterion.

Isolated longitudinally extensive transverse myelitis (LETM) extending over three or more vertebral segments without optic neuritis is now thought to be an early or limited form of NMO in some patients. In one study, NMO-IgG

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Figure 1 Diagnosis of neuromyelitis optica and longitudinally extensive transverse myelitis. (A) T2-weighted (left) and T1-weighted (right) sagittal MRI of the cervical spinal and upper thoracic cord in a patient with NMO, showing longitudinal extensive transverse myelitis with swelling, necrosis and linear gadolinium enhancement. Image kindly provided by Dr Anna Pichiecchio, Neurological Institute “C. Mondino”, Pavia, Italy. (B) Typical immunofluorescence pattern found on formalin-fixed adult mouse brain tissue immunostained with NMO-IgG1-positive serum. The staining pattern shows that the autoantigen is localized around the microvasculature. (C) Antibodies to AQP4 in NMO identified by an assay that employs EGFP-tagged AQP4 solubilized from transfected human embryonic kidney cells; results are expressed as fluorescence units immunoprecipitated by the sera.89 (D) Immunocytochemistry showing activation of complement C3b deposits (red) on the surface of EGFP–AQP4-transfected human embryonic kidney cells incubated in serum from a patient with NMO. Abbreviations: AQP4, aquaporin-4; EGFP, enhanced green fluorescent protein; MS, multiple sclerosis; NMO, neuromyelitis optica.

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was detected in over 50% of patients with LETM.6 NMO-IgG is also detectable in a subset (10–20%)35,36 of patients with recurrent optic neuritis and might predict conversion into NMO in some of these individuals.36

Differential diagnosisThe most important differential diagnosis of NMO is MS. NMO has been repeatedly reported in patients with systemic connective-tissue diseases (CTDs) such as lupus ery thematosus, Sjögren’s syndrome or antiphospholipid syndrome.11,37,38 NMO and CTDs can, however, occur independently, and NMO can associate not only with CTDs, but also with a wide variety of other autoimmune diseases.10,11,13 Furthermore, recent reports,8,38 as well as unpublished data from our laboratory, indicate that AQP4-Abs are as common among patients with both CTD and NMO as they are among uncomplicated NMO cases, but they are not present in patients with CTD without CNS involvement or with CNS involvement other than NMO. It is, therefore, reasonable to assume that NMO and CTD repre-sent independent pathological entities, although they might arise in the same patient owing to a general predisposition to autoimmune disease. The majority of patients with longitudinally non-extensive transverse myelitis (i.e. lesions extending over fewer than three segments) do not harbor antibodies to AQP4.8,39

IMMUNOLOGICAL FEATURES OF NEUROMYELITIS OPTICANMO-IgGThe diagnosis of NMO, and its distinction from MS, has been facilitated considerably by the discovery of NMO-IgG. NMO-IgG immuno-stains microvessels in the white and gray matter as well as the pia mater on formalin-fixed adult mouse cerebellum, brain and spinal cord tissue (Figure 1B). Cerebellar tissue shows the most prominent capillary staining and is preferentially used for immunofluorescence-based detection of NMO-IgG. Testing for NMO-IgG has a reported sensitivity of 58–76% and a specificity of 85–99% for NMO.6,9,10,17 Although a positive result is often present at disease onset, a negative result does not rule out a diagnosis of NMO.

Aquaporin-4 antibodiesDual immunolabeling of mouse brain sections with NMO-IgG antibodies and antibodies specific for the endothelial marker factor VIII,

the reactive-astrocyte marker glial fibrillary acidic protein (GFAP), or the extracellular matrix protein laminin, has suggested that the main target antigen is in astrocytes located adja-cent to the blood–brain barrier (BBB).6 AQP4 is a water channel located in astrocytic foot proc-esses, as well as in the kidney and stomach, and NMO-IgG staining colocalized with antibodies to AQP4 in tissue sections from both brain and extra cerebral organs.7 Studies in Aqp4 knockout mice and AQP4-transfected cell lines confirmed AQP4 as a target structure in NMO-IgG-positive patients with NMO.7

Further studies on AQP4-transfected cell lines have demonstrated the presence of AQP4-Abs in 91% of Japanese patients with NMO and in 84% with LETM.40 Our results have confirmed that a cell-based or a novel fluorescence-based immuno precipitation assay that employs recombi nant human AQP4 coupled to enhanced green fluorescent protein (Figure 1C) has higher sensitivity and specificity for NMO than does the NMO-IgG assay.89 The increased sensi-tivity of AQP4 assays compared with NMO-IgG testing suggests that AQP4 is the main—or possibly the only—target for NMO-IgG.

AQUAPORIN-4 LOCALIZATION AND FUNCTIONAQP4 is an osmosis-driven, bidirectional water channel that belongs to the subfamily of strict mammalian aquaporins, which are impermeable to anions and glycerol. The protein is expressed in two isoforms: M1-AQP4 (323 aa, 34 kDa) and M23-AQP4 (301 aa, 32 kDa). The protein mono-mers consist of six membrane-spanning α-helices and two pore helices that determine the channel’s selectivity for water molecules (Figure 2A). Both termini are located intra cellularly.41–45 Like other aquaporins, AQP4 forms homotetramers

Box 1 Revised diagnostic criteria for neuromyelitis optica.10

Optic neuritis

Acute myelitis

Plus at least two out of the following three supporting criteria:■ Brain MRI at onset not meeting Paty’s criteria for multiple sclerosis92

■ Contiguous lesion extending over three or more vertebral segments on spinal cord MRI

■ NMO-IgG seropositive status

Abbreviation: NMO, neuromyelitis optica.

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with four water-permeable pores per tetramer. M23-AQP4 tetramers and M1-AQP4 tetramers can form mixed arrays on the cell surface, although the M1 tetramers tend to disturb the array formation thereby limiting the size of the arrays.

AQP4 is found on all surfaces of astrocytes, but they occur at the highest concentration in the domains of the perivascular and peripial end-feet that are in direct contact with the basal lamina of the endothelium and pia mater, respectively (Figure 2B).46 AQP4 has also been

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Figure 2 The structure and localization of aquaporin-4. (A) Each AQP4 monomer consists of six membrane-spanning α-helices with both termini located intracellularly. Water selectivity depends on two pore helices and their highly conserved Asn–Pro–Ala motifs. Like other aquaporins, AQP4 forms homotetramers (not shown). (B) Polarization of AQP4 within the astrocytic end-feet at the glial–pial interfaces of the subarachnoid space and the Virchow–Robin space, and the glial–endothelial interface of arteries and arterioles. Astrocytic processes, together with collagen fibers of the subpial space, form the glia limitans externa that seals the brain surface, and form a dense sheath around capillaries, thereby constituting an integral part of the blood–brain barrier. (C) AQP4 is connected to the dystrophin-associated protein complex, which connects the astrocytic cytoskeleton via α1-syntrophin with the basal lamina and is mainly responsible for the polarization of AQP4 at the astrocyte end-feet. Abbreviations: AQP4, aquaporin-4; α-DG, α-dystroglycan; β-DG, β-dystroglycan; DP71, dystrophin variant DP71; PDZ, PDZ domain of α1-syntrophin; SXV, Ser-X-Val domain of aquaporin-4; Syn, α1-syntrophin. Permission for panel A obtained from Nature Publishing Group © Badaut J et al. (2002) J Cereb Blood Flow Metab 22: 367–378. Permission for panel C obtained from Nature Publishing Group © Amiry-Moghaddam M and Ottersen OP (2003) Nat Rev Neurosci 4: 991–1001.

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detected in ependymal-cell membranes, but not in neurons, oligodendrocytes or choroidal epithelial cells.43 Recent data suggest that AQP4 is more prominently expressed within optic nerves, brainstem and gray matter of the spinal cord than in the supraventricular white matter, and this correlates with the preferred lesion sites in NMO.47,48 The periventricular regions and the hypothalamus are also considered to be sites of high AQP4 expression. Outside the CNS, AQP4 is present in the distal collecting tubes of the kidney and parietal cells of the stomach. No appreci-able renal or parietal cell dysfunction has been reported, however, in α1-syntrophin-deficient (α-Syn-/-) mice, which show a selective loss of peri-vascular AQP4; these findings suggest redundancy of AQP4 in these tissues.49

AQP4 is co-expressed with the potassium channel Kir4.1 (IRK10). The polarization of both channels within astrocytic foot processes and the high density of AQP4 are mediated mainly by agrin, a proteoglycan within the basal lamina that binds to the α-dystroglycan compo nent of the dystrophin–dystroglycan complex. This complex in turn anchors AQP4 to the plasma membrane via α1-syntrophin (Figure 2C).50 Intriguingly, these interactions are highly reminiscent of those that anchor acetylcholine receptors at the neuromuscular junction.51

Little is known about the consequences of disrupting AQP4 function. Astrocytes transport water, derived from glucose metabolism within neurons, to the perivascular space, from where it is further drained via the CSF and the lymphatic system. In α-Syn-/- mice, mild swelling of astro-cytic end-feet has been reported, and the edema that usually occurs after hypo-osmotic stress or ischemia was reduced in comparison with control mice.52 By contrast, the severity of arti-ficially induced seizures was increased, probably as a result of disturbed potassium clearance.53

DISEASE MECHANISMS IN NEUROMYELITIS OPTICAThe existence of a highly specific antibody in NMO and LETM strongly suggests that humoral immunity has a substantial role in these condi-tions. To explore the function of autoantibodies in NMO, it is important to consider the occur-rences of other autoimmune diseases in patients with NMO and the associations of human leukocyte antigens (HLA) with this disease. The characteristics of the antibodies, the pathology and immunopathogenic mechanisms, treatment

Box 2 Summary of circumstantial evidence for a pathogenic role for aquaporin-4 antibodies in neuromyelitis optica.a

General features of the AQP4 antibody (AQP4-Ab)■ Highly specific for NMO (up to 98%)

■ Detectable in the majority of patients with NMO (68–91%)

■ Binds to a cell-surface antigen

■ Binds to the extracellular domain of AQP4

■ AQP4-Ab titers correspond with extension of spinal cord and brain lesions, although AQP4-Ab is not simply a marker of fulminant CNS involvement93

Pathogenic potential and mechanisms■ AQP4-Ab belongs to IgG subclass 1

■ AQP4-Ab has been shown to activate complement after binding to membrane-bound AQP4; this is in accordance with histopathological data that demonstrates complement deposits and lytic complement activation (membrane-attack-complex formation)

■ Marked loss of AQP4 within NMO lesions, partly without loss of glial fibrillary acidic protein, indicates that AQP4 loss might be a primary event

■ Foci of AQP4 loss correspond to sites of immunoglobulin and complement deposits

■ Similar perivascular rim and rosette pattern of normal AQP4 expression and deposits of immunoglobulin and complement are observed

■ Lack of reactive AQP4 expression in the periplaque region (as seen in multiple sclerosis), suggests a targeted response against AQP4

■ Distribution of AQP4 throughout the normal CNS is compatible with preferential distribution of NMO lesions

■ Non-multiple-sclerosis-like brain MRI lesions correspond to sites of high AQP4 expression

Treatment responses■ Preliminary data suggest a beneficial effect

of treatments targeted towards B cells or antibodies, such as rituximab (anti-CD20) and plasma exchange

■ Preliminary data indicate a correlation between AQP4-Ab titers and disease activity (S Jarius, unpublished data)

aThese observations are supportive of a potentially pathogenic role for AQP4 antibodies. More-conclusive evidence should come from passive transfer and active immunization models. Abbreviations: AQP4, aquaporin-4; NMO, neuromyelitis optica.

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responses, and animal models of disease must also be considered. In this section, we will discuss some of the features that relate to the immuno-pathogenesis of NMO, as summarized under three main headings in Box 2. These features are reminiscent of considerations relevant to the pathogenesis of myasthenia gravis,54 the prototypical antibody-mediated disease, but the conclusions have to take into account the probable location of AQP4 behind the BBB.

Autoimmunity and human leukocyte antigen associationsLittle is known about the genetic background of NMO.55 In contrast to MS, NMO is not positively associated with the DR2-associated DRB1*1501 allele. In Asian patients with OSMS, a positive association with DPB1*0501 and a negative association with DPB1*0301 have been described.57,58 These findings have recently been challenged, however, by a study that suggests that the association of DPB1*0501 with OSMS might be attributable to over-representation of the DPB1*0301 allele among patients with

conventional MS.59 Moreover, a study from the UK did not find any association with DPB*0501 in white patients with NMO.60 Nevertheless, there is some evidence of high frequencies of occurrence of other autoimmune disorders and positive family histories of autoimmune diseases among patients with NMO.10,13 Non-organ-specific autoantibodies are present in 50% of patients with NMO.10

Immunopathology Acute NMO lesions are dominated by edema and necrosis, whereas chronic lesions are character-ized by gliosis and atrophy. Lesions usually extend over three or more vertebral segments and predominantly affect the central parts of the spinal cord. Spinal cavities can occur as a result of necrosis.61,62 On histological examination, extensive demyelination and substantial axonal damage (e.g. swelling, spheroid formation and/or reduction of axonal density) can be detected.5 Both white and gray matter are involved. Signs of remyelination are rare. Inflammatory infil-trates mainly consist of cells of the macrophage–microglia lineage, along with neutro phil and eosinophil granulocytes; CD3+CD8+ T lympho-cytes are seen in frequently. AQP4 is expressed predominantly around blood vessels, and, in NMO, the peri vascular immuno globulin and complement deposits that surround blood vessels are seen in a distinctive rim or rosette fashion (Figure 3A–C). This suggests that the AQP4-Abs can access and target their antigen.5 This localization is different from that described for type II MS lesions, in which the comple-ment deposits are found at the lesion edge, spatially associated with oligodendrocytes, as well as within macrophages (Figure 3D).63 As already described by Devic in 1895, signs of hyalinization and fibrotic thickening of vessel walls are found in NMO lesions, as is increased vessel density.2,5,64 Typical features of necro-tizing vasculitis (e.g. fibrinoid necrosis and/or granulo cyte infiltrates in vessel walls), however, are usually absent.5

Periventricular hypothalamic lesions affecting the area postrema (with or without associated endocrinopathies) have recently been described in some AQP4-Ab-positive patients.12,27 These areas are not only characterized by a high density of astrocytes and strong AQP4 expres-sion, but might also function as an interface between the immune system and the brain.65 Like other circumventricular organs, the area

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Figure 3 Complement deposits at sites of aquaporin-4 loss in neuromyelitis optica but not in multiple sclerosis. Neuromyelitis optica lesions are characterized by (A) a distinct perivascular rim or (B) a rosette or mesh pattern of complement C9neo deposition, which corresponds well to (C) perivascular aquaporin-4 (brown) expression in the healthy CNS. (D) By contrast, in type II multiple sclerosis lesions, complement deposits (C9neo, brown) are found within macrophages (arrowheads) and on oligodendrocytes at the lesion edge (not shown), but not around vessels (arrow). Permission obtained from Guarantors of Brain © Lucchinetti CF et al. (2002) Brain 125: 1450–1461 (A,B) and Roemer SF et al. © Roemer SF et al. (2007) Brain 130: 1194–1205 (C,D).

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postrema lacks a BBB and has, accordingly, been implicated in antibody and immune-cell traf-ficking in conditions such as experimental auto-immune encephalomyelitis, a rodent model of MS.66 Moreover, the periventricular organs have been implicated in regulation of osmolarity and cerebral blood flow.47,67

What determines the characteristic cellular infiltrates?The infiltrates seen in patients with NMO are mainly, as mentioned above, macrophages–microglia, neutrophils, and, somewhat surpris-ingly, eosinophils. Immunohistochemistry shows marked expression of the most important eosino phil chemokine receptor, CCR3, signs of eosinophil degranulation in NMO lesions,5 and increased CSF levels of a variety of chemo-kines that are potent eosinophil attractants.68 Eosinophils are considered to be important components of type 2 helper T cell (TH2)-mediated humoral immunity through their production of interleukin (IL) 4, and TH2 cells are known to express CCR3, rendering them potentially prone to recruitment by eotaxins. Ishizu et al. reported intrathecal activation of the IL-17–IL-18 axis in NMO,69 which could explain the involve-ment of neutrophil granulocytes. It is unclear how these cells are involved in the patho-genesis of NMO, and whether they relate to an antibody-mediated mechanism.

Aquaporin-4 antibodies and their potential pathogenicityIn myasthenia gravis, the antibodies to acetyl-choline receptors bind to extracellular domains of the antigen and are predominantly IgG1, are of high affinity, and can activate complement. Similarly, the AQP4-Abs bind to the extra cellular domains of AQP4 expressed in un permeabilized human embryonic kidney cells,7,70 which in di cates that they have the potential to bind in vivo (if they manage to get beyond the BBB). These antibodies are mainly IgG1 (as illustrated for NMO-IgG in Figure 1B), with some IgG4, and can activate complement, as demonstrated by deposition of complement C3b (Figure 1D),89 or C9neo,71 on the cell surface in the presence of fresh human serum as a source of complement. The tendency of AQP4 to form arrays is likely to increase the affinity of the interaction between the IgG and the cell surface, and this might be important in the immunopathology of NMO. Our research group found that NMO serum

IgG binds detergent-solubilized AQP4 (as in Figure 1C) with relatively low affinity, probably because the detergent-extracted antigen behaves as a single tetramer.89

Loss of aquaporin-4A marked loss of AQP4 immunoreactivity within spinal cord lesions in patients with NMO, in dependent of disease stage, has recently been demonstrated (Figure 4A).47,48,72 Importantly, foci of AQP4 loss in NMO corresponded well to the sites of perivascular immunoglobulin and complement activation,47 in contrast to MS in which AQP4 loss is restricted to in active plaques (Figure 4B–D). In MS, AQP4 expres-sion is increased in the center of actively demyelina ting or remyelinating lesions, as well as in the white matter surrounding such lesions (Figure 4B–C).47,73

In the same study, the authors described two histological types of NMO lesion: a demyelinat ing cavitary type predominant in the

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Figure 4 Aquaporin-4 expression in neuromyelitis optica and multiple sclerosis. All images show spinal cord lesions. (A) In neuromyelitis optica, a marked and stage-independent loss of AQP4 (brown) is found within spinal cord lesions, although it is retained in the periplaque white matter. (B,C,D) AQP4 immunoreactivity (brown) is stage-dependent in multiple sclerosis. In the case of active demyelinating lesions (B), AQP4 expression is increased in the adjacent cortical gray matter (arrow) and periplaque white matter. In active remyelinating lesions (C), there is diffusely increased expression in both the center of the lesion and the periplaque region. In chronic inactive lesions (D), there is complete loss of AQP4. Abbreviations: AQP4, aquaporin-4; PPWM, periplaque white matter. Permission obtained from Roemer SF et al. © Roemer SF et al. (2007) Brain 130: 1194–1205.

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spinal cord and optic nerves (type A), and a highly inflammatory type without demyelina-tion or axonal pathology found in the brainstem and the spinal cord (type B).47 In view of the finding that brainstem lesions often remain clin-ically silent in NMO and are reversible in some patients, as demonstrated by MRI, the authors speculate that type B lesions might reflect revers-ible functional impairment of astrocytic water flux caused by IgG-mediated blocking of AQP4. These findings were confirmed by a study from Japan, which described relatively preserved myelinated fibers in acute inflammatory, actively demyelinating and even chronic active NMO lesions.48 The preservation of myelin despite a complete loss of AQP4 immuno reactivity in some NMO lesions (contrasting with the marked loss of myelin proteins and increased AQP4 expression in MS) suggests that demyelina-tion might be secondary to astrocyte damage in NMO (Figure 5A,B).

One study reported that the loss of AQP4 immunoreactivity in spinal cord lesions was paralleled by a loss of GFAP in some NMO

lesions from an early stage of the disease.48 This observation further distinguishes NMO from MS lesions, which are characterized by strong reactive astrogliosis and, consequently, increased GFAP expression.48,74,75 The limited extent of astrogliosis in NMO is consistent with the proposed immune attack against astrocytes, as well as with recent findings that indicate a role for AQP4-Ab in astroglial migration.76 Interestingly, Roemer and colleagues observed normal GFAP staining in type B lesions; this strengthens the case for AQP4 loss in these lesions preceding astrocyte loss (Figure 5C,D).47

Overall, there is circumstantial evidence for an autoimmune pathology with loss of AQP4 in NMO lesions, along with associated immune-complex deposition, but the pathogenic mecha-nisms that underly tissue edema and necrosis and lead to irreversible loss of function are not clear.

Responses to treatmentThe treatment of NMO has been reviewed else-where,77,78 and here we will present only the evidence that points to an autoimmune patho-genesis in the disease. Acute symptoms respond to short courses of high-dose intra venous corticosteroids in up to 80% of patients,11 and, more importantly, case reports and small case series have indicated that plasma exchange or lymphocytapheresis is beneficial.11,79–82 A study of plasma exchange in patients with severe CNS demyelinating disorders reported functional improvement in 7 out of 10 patients with NMO.83

Recently, rituximab, a chimeric anti-CD20 monoclonal antibody that depletes mature and precursor B cells, was shown to improve NMO-related disease activity and disability in seven out of eight patients when used as primary or secondary maintenance therapy.84 This points more directly to a humoral immuno pathogenesis in NMO. Furthermore, mitoxantrone, which targets predominantly B cells and macro-phages,85–87 seems to be useful as a disease-modifying agent in NMO. Overall, these reports support an autoimmune—possibly humorally mediated—pathogenesis in the disease.

Passive transfer and active immunizationThe final proof of an antibody-mediated mecha-nism for NMO would be achieved by demon-strating passive transfer of the disease, but this has not yet been reported. In view of the fact

100 μm

200 μm

100 μm

200 μm

A B

C D

Figure 5 Aquaporin-4 loss seems to be primary rather than secondary to astrocyte loss in neuromyelitis optica. (A) Myelin (blue) is relatively preserved in some neuromyelitis optica lesions despite (B) marked AQP4 loss, which suggests that demyelination is secondary to AQP4 loss. Moreover, (C) the reactive-astrocyte marker glial fibrillary acidic protein (brown) is expressed despite AQP4 loss (D) in some lesions, which suggests that AQP4 loss occurs before astrocyte death in neuromyelitis optica. Abbreviation: AQP4, aquaporin-4. Permission obtained from Roemer SF et al. © Roemer SF et al. (2007) Brain 130: 1194–1205.

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that NMO-IgG was first defined by immuno-fluorescence on mouse tissue, this species should be a suitable model to use for such studies. AQP4-Abs seem to be predominantly located in the serum rather than the CSF; therefore, the disease would need to be modeled by systemic administration of IgG, rather than intrathecal application. It will be interesting to see whether peripherally injected NMO-IgG can access AQP4, whether the AQP4-Abs themselves damage the BBB by altering astrocyte function, and whether those areas of the brain where the BBB is not present, such as the periventricular organs and hypothalamic regions, are particularly vulnerable to complement-mediated damage.

Summary: evidence supporting a role for aquaporin-4 antibodiesBox 2 and Table 1 summarize some of the data relating to the pathogenesis of NMO compared with MS. Our current knowledge regarding AQP4-Ab fits well into the concept of NMO as a humorally mediated autoimmune disease. Many of the serum reactivities of unknown pathogenic importance associated with neurological condi-tions—particularly paraneoplastic dis orders—are directed against intracellular targets, but AQP4 is located on the plasma membrane, and is, therefore, directly accessible to antibodies. The loss of AQP4 within spinal cord lesions, a finding that distinguishes NMO from MS,47,72,88 strongly suggests that the antibodies can target their antigen in vivo. A primary response against

AQP4, as opposed to a secondary loss of AQP4 through astrocyte damage, is also favored both by the finding that GFAP is relatively preserved in some lesions, and by the lack of AQP4 loss in the periplaque white matter,47 although the channel might be lost in other regions owing to tissue necrosis.48 Importantly, foci of AQP4 loss were demonstrated to coincide with sites of intense vasculocentric immune-complex deposition, further supporting a possible role for AQP4-Ab as the initiator of NMO lesions. It is noteworthy that the MRI lesions typical of NMO correlate well with the normal distribution of AQP4.27,47,48

Further lines of evidence that AQP4-Ab could be involved in NMO pathogenesis come from a recent study that reported a positive correla-tion between AQP4-Ab titers during relapse and longitudinal extension of spinal cord lesions (and possibly between AQP4-Ab titers and severity of optic nerve and brain involve-ment). In addition, observations from this study show that AQP4-Ab titers seem to correlate with clinical improvement and stabilization.40

Many questions remain unresolved. For instance, intralesional immunoglobulin deposits, as demonstrated by immuno histochemistry, consist mainly of IgM, whereas AQP4-Ab seems to be largely of the IgG class (both in serum and in CSF).71,89 Moreover, it is not well under-stood why the antibody causes tissue damage in the CNS but not in the kidneys, the stomach or other organs in which the BBB would not

Table 1 Comparison of immunopathological features of neuromyelitis optica and multiple sclerosis.

Neuromyelitis optica Multiple sclerosis

White and gray matter involvement Predominant white matter involvement

Striking edema Inflammatory lesion

Necrosis Necrosis not striking

Cavitations No cavitations

Relatively preserved myelin in some lesions Severe demyelination

Axonal damage Axonal damage

Leukocyte infiltrates are mainly neutrophils and eosinophils rather than T and B lymphocytes

Leukocyte infiltrates are mainly T and B lymphocytes rather than neutrophils

Loss of aquaporin-4 in all lesions Upregulation of aquaporin-4 in active lesions

Loss of glial fibrillary acidic protein in most lesions Increased glial fibrillary acidic protein staining

Complement and IgM–IgG deposits around blood vessels Less-marked complement deposits within macrophages and at the lesion edge on oligodendrocytes/myelin (in type II multiple sclerosis lesions)

Vascularity increased Increased vascularity uncommon

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provide any protection. Similarly, it is unknown why inflammation in NMO is restricted mainly to the optic nerves and spinal cord, despite AQP4 being expressed throughout the entire CNS. Differential accessibility of AQP4 in vivo, and regional differences in the spatial distri-bution and density of the AQP4 epitopes that determine cross-linking and complement acti-vation (as seen in other autoimmune diseases),90 might explain this discrepancy.7,47,48 The lack of substantial disease in AQP4-deficient mice suggests that AQP4 is redundant, and that NMO reflects the resulting inflammation rather than loss of AQP4 function. Finally, the presence of high AQP4-Ab titers during remission in some patients with NMO renders it unlikely that the presence of AQP4-Ab is sufficient to cause clinical disease; additional prerequisites (e.g. disturbance of the BBB and/or T-cell involvement) might be required.

SERONEGATIVE NEUROMYELITIS OPTICASome patients are negative for NMO-IgG or AQP4-Ab even when the most sensitive assays are used, and attempts to identify novel anti-gens are in progress.91 The sensitivity of some assays could probably be improved further, although the use of human AQP4 expressed at a high concentration in cell lines, as by Takahashi et al.40 and by ourselves,89 is likely to be optimal for detection of the antibodies. It will be equally important to test samples taken during relapse and before treatment, as these are more likely to test positive for the antibodies.89

CONCLUSIONS AND FUTURE PROSPECTSThe recent observation of a distinct immuno-pathology in patients with NMO and the subsequent detection of AQP4-Ab as a unique serological biomarker have substantially advanced our understanding of NMO, and have raised the possibility of making a clearer distinc-tion at early disease stages between patients with NMO and patients with spinal cord and optic nerve demyelination associated with MS or other diseases. Longitudinal AQP4 antibody measure-ments will establish the relationship between antibody levels and clinical status. More-detailed immunopathology, and active immunization and passive transfer experiments, will be needed to help elucidate the precise role of AQP4-Ab in NMO.

KEY POINTS■ Neuromyelitis optica (NMO) is an inflammatory

disorder of the CNS of putative autoimmune etiology that predominantly affects the spinal cord and optic nerves

■ NMO is histologically characterized by extensive demyelination and substantial axonal damage; the presence of IgG and complement deposits suggests a humoral pathogenesis

■ Recently, a new serum reactivity (called NMO-IgG), characterized by binding of IgG to structures adjacent to the microvasculature and pia mater, has been detected in patients with NMO

■ Aquaporin-4, the most abundant water channel in the CNS, has been identified as the target antigen of NMO-IgG

■ Indirect evidence from immunobiological and histological studies suggests an important role for NMO-IgG/AQP4-Ab in the pathogenesis of NMO

■ These new findings facilitate the diagnosis of NMO and might soon translate into new therapeutic approaches

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AcknowledgmentsThe work of S Jarius was supported by a fellowship from the European Neurological Society. The authors are very grateful to Professor Margaret Esiri for helpful comments on the manuscript, and to Dr Isabel Leite and Dr Saiju Jacob for allowing us to show unpublished data.

Competing interestsA Vincent declared associations with the following companies: Athena Diagnostics and RSR Ltd. See the article online for full details of the relationships. The other authors declared no competing interests.

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Mechanisms of Disease: aquaporin-4 antibodies in neuromyelitis optica