Intracerebral immune complex formation induces inflammation in the brain that depends on Fc receptor interaction
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In this study, we investigate the underlying mechanisms of antibody-mediated inflammation in the brain. We show that immune complexes formed in the brain parenchyma generate a robust and long-lasting inflammatory response, characterized by increased expression of the microglia markers CD11b, CD68 and FcRII/III, but no neutrophil recruitment. In addition to these histological changes, we observed transient behavioural changes that coincided with the inflammatory response in the brain. The inflammatory and behavioural changes were absent in Fc-gamma chain (Fcγ)-deficient mice, while C1q-deficient mice were not different from wild-type mice. We conclude that, in the presence of antigen, antibodies can lead to a local immune complex-mediated inflammatory reaction in the brain parenchyma and indirectly induce neuronal tissue damage through recruitment and activation of microglia via Fcγ receptors. These observations may have important implications for the development of therapeutic antibodies directed against neuronal antigens used for therapeutic intervention in neurological diseases.
KeywordsFc receptor Antibody Immunotherapy Microglial activation
An inevitable consequence of an ageing population is an increased incidence of neurodegeneratative diseases, such as Alzheimer’s disease, Parkinson’s disease and age-related macular degeneration. Recent experimental and clinical studies have provided evidence for both innate and adaptive immune activation in the pathogenesis of these debilitating disorders. For example, microglia activation is typically associated with any neuropathology and there is increasing evidence that (auto)-antibodies against brain-reactive antigens are associated with clinical symptoms [11, 28]. Genome wide association studies (GWAS) in AD  and AMD  provide further evidence for the involvement of the immune system in disease pathogenesis. The young and healthy CNS is an immune privileged site where immune surveillance and inflammation is tightly regulated [4, 15]. The presence of an intact blood–brain barrier (BBB) combined with the unique microenvironment of the brain, ensures that the reactions to an inflammatory challenge, such as endotoxin (LPS) or cytokines, are attenuated . We and others have shown that the healthy CNS parenchyma is able to modify leucocyte responses to acute injury [2, 3], but it is less clear if, and how, the brain controls antibody-mediated responses, and whether these responses are altered under neuroinflammatory conditions or age-related pathology. Antibodies may mediate tissue damage when they form immune complexes and recruit cytotoxic effector cells, such as macrophages via their Fcγ receptors (FcγRs) or by activating complement . The interaction with FcγRs stimulates cell signalling in the effector cell that ultimately results in phagocytosis and/or release of inflammatory or cytotoxic mediators. These responses are well described in peripheral tissues, using the (reversed) Arthus reaction, a well accepted experimental model of antibody-mediated inflammation . In the presence of an intact BBB, IgG is only present in the healthy brain at very low levels relative to plasma levels  and the effector cells, such as microglia and perivascular macrophages, express detectable but low levels of FcγRs . However, expression of FcγRs is enhanced on microglia following treatment with IFN-γ, TNF-α and LPS in vitro , after intracerebral injection of LPS  and, as we have recently shown, during experimental chronic neurodegeneration . Despite these observations we have limited knowledge of the consequences of immune complex formation in the CNS, the associated inflammatory response and the function of the different FcγRs in the CNS. The growing incidence of neurodegenerative conditions in the human population, and the interest in the use of antibody-based immunotherapy to treat these diseases, highlights the need to understand the possible consequences of antibody-mediated inflammation in the CNS parenchyma.
Davidoff et al.  were the first to describe antibody-mediated inflammation in the brain and showed that the Arthus reaction in the rabbit brain resembled that seen in the skin and other peripheral tissues. However, it is likely that the relatively crude techniques used in this study significantly disturbed the unique vasculature and microenvironment of the brain making it difficult to interpret the results. More recently, Lister and Hickey  reported that immune complexes can be formed in the microvasculature of the brain, resulting in complement activation, increased microvascular permeability and leucocyte adhesion. However, this study was restricted to the role of immune complexes in the meningeal compartment and not in the brain parenchyma.
The aim of the current study was to investigate the acute and long-term consequences of immune complex formation in the brain parenchyma, using a model antigen widely used in the study of peripheral antibody-mediated responses. We show that immune complex formation in the brain parenchyma results in neuroinflammatory and behavioural changes that depend on FcγR interactions. Apart from further understanding of antibody-mediated responses in the brain in general, our study provides insight into the complications reported following anti-Aβ immunization, such as micro-haemorrhages and increased cerebral amyloid angiopathy (CAA) [6, 46, 47].
Materials and methods
BALB/c mice were obtained from Charles River (Margate, UK) and bred and maintained in local facilities. Fcγ chain deficient (Fcγ−/−) originally described by Takai et al.  were obtained from The Jackson Laboratory and back crossed onto a BALB/c background. C1q-deficient (C1q−/−mice were obtained from Dr Aras Kadioglu (Leicester, UK) with permission from Professor Marina Botto (London, UK) . Animal experiments were carried out with approval from the local Committee for Ethics at the University of Southampton and were performed under a Home Office license.
8 week old BALB/c mice were immunized against ovalbumin (OVA) by intraperitoneal injection of 50 μg OVA (Sigma) in the presence of Alum (1:1 ratio, Alum imject, Pierce). Mice were boosted three times (2, 4 and 6 weeks) by intraperitoneal injection of 100 μg OVA in saline. Three days after the last OVA injection, OVA was microinjected into the striatum.
Immune complex formation
Immune complex-mediated inflammation was initiated by a cerebral injection of OVA in OVA-immunized or non-immunized control animals. Mice were anaesthetized by intraperitoneal injection of 0.1 ml/5 g body weight Avertin (2,2,2 tribromoethanol in tertiary amyl alcohol) and placed in a stereotaxic frame (Kopf Instruments, Tujunga, CA, USA). OVA (10 μg in 1 μl) was injected into the striatum (bregma +1 mm anterior, lateral +1.5 mm, 2.5 mm deep) by a minimally invasive technique using a fine glass micropipette with a diameter of <50 μm (Sigma). Tissue was collected after 24 h, 3 days, 7 days, 14 days and 28 days. These time points were chosen based on the kinetics of immune complex-mediated inflammation in peripheral organs  or microglial activation in the brain parenchyma following LPS challenge . Under terminal anaesthesia, blood samples were collected by cardiac puncture, and after transcardial perfusion using heparinized saline, brains and spleen were removed and snap frozen in OCT embedding medium. For immunohistological examination and quantification studies, brains were sectioned in a coronal plane on a cryostat (Leica 17–20).
Serial sections of brain, 10 μm thick, were air-dried and fixed in cold ethanol for 10 min at 4 °C, and stained for the presence of immune complexes. Rabbit anti-OVA (Sigma, UK) was used to detect the antigen OVA and complement activation was identified using antibodies against C3 (FITC conjugated rabbit anti-C3, Cappel). Mouse IgG was identified using FITC labelled F(ab′)2 fragments of goat anti-mouse IgG (Sigma, UK). Phenotypic changes in macrophages were assessed using rat anti-mouse F4/80 (serotec), CD11b (5C6, Serotec), CD68 (FA11, Serotec), MHC class II (Bioscience) and CD16/CD32 (FCR4G8, FcRII/III, Serotec). The presence of neutrophils was assessed with MBS-1, an in-house produced polyclonal antibody, generated as described elsewhere . The presence of platelets was assessed with a rat anti-mouse gpII1/IIIb mAb (CD41, Serotec) and T cells using rat anti-mouse CD3 (KT3, Serotec). Biotinylated secondary antibodies and HRP-conjugated streptavidin were from Vector (UK) and the chromogen substrate DAB from Sigma (UK). Alexa Fluor 488 or Alexa Fluor 546 conjugated secondary antibodies were obtained from Invitrogen (Molecular Probes, Oregon, USA). Mounted sections were cover-slipped using Vectashield (Hard set, with DAPI, Vector, UK). The intensity of the macrophage markers CD11b, F4/80 and MHC class II was quantified using the Leica analysis software on a Leica microscope. The expression level of macrophage markers was quantified by taking four images at 10× magnification per injected hemisphere (field). The total number of pixels of DAB positive staining per field was recorded. Data was analysed by One-way ANOVA followed by Dunnett’s post hoc test. p < 0.05 was considered significantly different.
OVA antibody ELISA
Sera from OVA-immunized mice was serially diluted onto OVA coated plates (10 μg/ml in PBS; maxiSorb, Nunc) followed by incubation with biotinylated horse-anti-mouse IgG (Vector, UK) for determination of total OVA-specific IgG levels. Subclasses were determined by IgG1- and IgG2a-specific antibodies (Serotec, UK). Binding of OVA-specific antibodies was detected by poly-streptavidin (Sanquin, The Netherlands) and visualized using TMB/H202 substrate (R&D systems, UK).
Assessment of behaviour
Circling behaviour was carried out in an opaque cylindrical bowl of 10 cm circumference, with a clearly labelled mid-point that was used as a reference as to how many times the mouse crosses the line and in which direction. Left and right turns were counted when the tip of the nose crossed this reference line, over a 1-min period. The left and right turning behaviour was measured at day −1 for baseline measurements and at day 1, 3, 7 and 14 after intracerebral OVA injection.
Quantification of immune activation markers and behavioural data was analysed by one-way analysis of variance (ANOVA) followed, if significant, by Dunnett’s post-test versus controls using Graphpad Prism software. Values were expressed as mean ± SEM. A p value <0.05 was considered to indicate statistical significant difference and n refers to number of animals per group.
Immune complexes in the brain parenchyma
Neuroinflammation in the absence of neutrophils
Differential role of complement and Fc receptors
In this study we have demonstrated that the formation of immune complexes in the brain parenchyma results in a localized neuroinflammatory response and associated behavioural changes. We show that immune complexes form in association with cerebral blood vessels of OVA-sensitized mice that have received an intracerebral OVA challenge. Immune complex formation results in increased expression of FcγRII/III, CD11b, F4/80, CD68 and MHCII on perivascular macrophages and microglia, observed from 3 days until 4 weeks after antigen challenge. At 24 h we detected platelets adhering to blood vessels, but neutrophils were not detected at any time point measured. We further showed that the interaction with FcγRs is critical for the induction of this inflammatory response, since mice lacking the γ-chain did not show the histopathological changes or altered rotation behaviour, despite similar levels of circulating OVA-specific IgG. Some characteristics of immune complex-mediated inflammation in the CNS are similar to those observed in skin or lung inflammation models, including extravasation of IgG, activation of the complement cascade and activation of macrophages, but it differs significantly in the kinetics and cellular components recruited.
The role of FcR in antibody-mediated neuroinflammation
The mechanisms of immune complex-mediated inflammation have been extensively studied in peripheral organs, such as the skin and the lung. It was shown that when antigen–antibody immune complexes are formed at vascular basement membranes in extracerebral sites, they trigger inflammation, characterized by oedema, recruitment of neutrophils, complement activation, and local tissue damage [10, 41]. It has been suggested that both the complement system and the activation of Fc receptors contribute to these inflammatory response [39, 40]. The effects of immune complex triggered inflammation in the brain and associated neurobehavioural consequences have only been sporadically reported in the literature. In 1932, Davidoff et al. reported that rabbits, sensitized against egg albumin showed a typical sterile inflammation characteristic for local anaphylactic symptoms, following intracerebral challenge with the same antigen. The pathological changes were similar to those described earlier in the skin , including tissue necrosis, oedema, haemorrhages and infiltration of leucocytes. The surviving rabbits developed behavioural changes, including tonic and clonic muscular contractions and rotating movements. These observations were the first to describe the devastating consequences of immune complex formation in the brain, but due to the relatively crude methodology and many fatalities, the results should be interpreted with care. Our study shows that there are important differences between immune complex-induced inflammation in the brain and other tissues, but highlight the similar role for FcγRs in the initiation of IgG-mediated inflammation and functional behavioural changes. Although components of complement appear less critical our study only used C1q−/− mice, and to rule out other factors of the complement system further studies are required. Previous studies using unilateral, intrastriatal injections of LPS have shown similar effects on microglia and circling behaviour . The molecular mechanisms underlying these changes include increased expression of MHCII, cytokines and iNOS in the substantia nigra and striatum, but whether a similar mechanism explains the behavioural changes in our model remains undetermined.
A limited number of studies looked at the mechanism underlying neuropathology following immune complex formation, but the role of FcγR is largely unknown. Schupf and William  showed that injection of preformed OVA-anti-OVA immune complexes into the hypothalamus of rats results in increased food intake. As the effect was not observed upon injection of immune complexes containing F(ab′)2 fragments, the authors concluded that the effects observed depend on complement activation. However, as F(ab′)2 fragments lack Fc, interaction with FcγRs, cannot be excluded. A more recent study using a model of neuromyelitis optica (NMO) also suggests a key role for complement in the mechanism of antibody-mediated inflammation in the CNS . Saadoun et al.  demonstrated that intracerebral injection of human IgG into mouse brain only induces pathology when co-injected with human complement. The pathology was characterized by infiltration of monocytes, but not granulocytes, and ipsiversive rotation behaviour, similar to our study. FcγRs display highest affinity to IgG of the same species  possibly explaining why human antibody alone did not induce pathology, while mouse antibodies in our study do so.
Initiation of antibody-mediated neuroinflammation
Immunohistochemical data show that immune complexes deposit in the brain parenchyma within 24 h after antigen challenge. We cannot rule out that the use of a micropipette for intracerebral OVA challenge induces BBB leakage. However, immune complexes were not observed solely in the injection site, but observed throughout the challenged hemisphere. In addition, intra-vitreous injections that do not damage the blood–retinal barrier (BRB) lead to a similar inflammatory response (unpublished observations), suggesting an alternative mechanism for increased IgG influx into the CNS. It is generally believed that circulating antibodies are restricted to enter the brain due to an intact BBB, but it has been shown that very low levels of IgG (~0.1 %) can gain access to the brain via the extracellular pathway . We show that mice used in our study have high serum titers of OVA-specific antibodies, and under these conditions, the low level of IgG that crosses the intact BBB is possibly sufficient to initiate the deposition of immune complexes. The perivascular space is likely the primary site of immune complex-induced inflammation as we find constitutive and then rapidly increased expression of FcγR on perivascular macrophages. It has also been suggested that activated T cells play a role the breaking the integrity of the BBB in models of auto-immune-mediated neurological diseases. Hu et al.  show that in the presence of antigen, activated T cells extravasate into ocular tissue, resulting in a monocyte recruitment and further breakdown of the BRB. Similar findings have been described in animal models of demyelination in the spinal cord , but high numbers (3–5 × 106) of T cells were needed to induce opening of the BBB. We detected small numbers of CD3+ T cells in our model, therefore, we cannot exclude a role for T cells in altering BBB permeability and increased IgG influx. However, CD3+ T cells were also observed in FcγR−/−, suggesting that increased microglial activation depends on interaction with FcγRs.
In the present study we show that OVA-anti-OVA immune complexes-induced expression of CD11b, CD68, MHC class II as well as marked expression levels of FcγRs on microglia. Similar results have been reported following intracerebral injection of anti-Aβ antibodies in APP transgenic mice . Humoral components have been implicated in the pathogenesis of neurodegenerative diseases although it is controversial as to what extent the antibodies are pathogenic or simply a consequence of the ongoing neurodegeneration . Engelhardt et al.  showed loss of cholinergic neurons following injection of IgG isolated from an AD patient. Similar observations were made using IgG derived from PD patients resulting in microglial activation and loss of hydroxylase (TH+) neurons. Intracerebral administration of PD-derived IgG results in perivascular inflammation, significant microglial activation and increased rotational behaviour . Interestingly, the effects on microglial activation were absent in FcγR−/− mice . Postmortem analysis of PD brain tissue shows similar histopathological changes as those observed in the animal models . These observations suggest that, apart from classic CNS autoimmune disorders, immune complexes may contribute to the pathogenesis and/or ongoing pathology of neurodegenerative diseases.
Implications for immunotherapy
There is growing academic and commercial interest in utilizing the power of antibodies to treat AD by vaccination against Aβ peptides  but immunotherapy targeting of brain antigens is not without risks. Histological examination of the brains of immunized humans reveals that, although immunization reduces the plaque load in the parenchyma , vascular Aβ deposits persist, leading to increased incidence of haemorrhages . Experimental models have shown that antibodies devoid of the Fc region, such as Fab′, F(ab′)2 and scFv antibodies, or de-glycosylated antibodies, which cannot engage effector systems, successfully remove Aβ from the brain without inducing haemorrhages [14, 33, 45]. Furthermore, increased expression of FcγR expression levels are reported following passive immunization with Aβ-specific antibodies in APP transgenic mice, which did not occur after deglycosylation of the therapeutic antibody [8, 48]. These observations suggest that antibodies facilitate in the removal of plaques, but their Fc regions can cause detrimental inflammatory reactions through interaction with perivascular macrophages and microglia. Another potential side-effect of immunotherapy is the solubilization of Aβ peptides from plaques that remain trapped in the perivascular drainage pathways, leading to worsening of cerebral amyloid angiopathy . We hypothesize that formation of immune complexes between Aβ peptides and Aβ antibodies and subsequent inflammation may partly explain increased CAA following immunotherapy. A better understanding of FcγRs and controlling FcγR function in the brain microenvironment will likely increase the success of immunotherapy for neurodegenerative diseases and reduce clinical setbacks experienced to date.
We thank the Alzheimer’s Research UK [pilot2006B to R.O.C.] and The Wellcome Trust [WT082057MA to J.L.T. and V.H.P.] for funding the work and we thank Sara Waters and Richard Reynolds for excellent technical assistance.
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