C3c and C4d were detected in all ischaemic lesions, suggesting activation via the classical ... MBL clone HYB 131-01, 131-10 and 131-11 (Antibody- shop ...
CLINICAL IMMUNOLOGY doi: 10.1111/j.1365-3083.2009.02253.x ..................................................................................................................................................................
In Situ Deposition of Complement in Human Acute Brain Ischaemia E. D. Pedersen*, E. M. Løberg , E. Vege , M. R. Dahaà, J. Mæhlen & T. E. Mollnes*
Abstract *Faculty of Medicine, Institute of Immunology, Rikshospitalet University Hospital, University of Oslo; Department of Pathology, Faculty of Medicine, Ullevaal University Hospital, University of Oslo, Oslo, Norway; and àDepartment of Nephrology, Leiden University Medical Center, ZA Leiden, The Netherlands
Received 30 January 2009; Accepted in revised form 2 March 2009 Correspondence to: E. D. Pedersen, Institute of Immunology, Rikshospitalet University Hospital, N-0027 Oslo, Norway. E-mail: elena.pedersen@ rr-research.no
Experimental animal models indicate that complement contributes to tissue damage during brain ischaemia and stroke, but limited data are available for a role of the complement in human stroke. We, therefore, evaluated whether acute ischaemia leads to complement activation in human brain. Indirect immunohistochemical staining was performed on paraffin-embedded, formalin-fixed human brain from 10 patients and 10 controls. Complement components C1q, C3c and C4d were detected in all ischaemic lesions, suggesting activation via the classical pathway. C9, C-reactive protein and IgM were detected in necrotic zones. Marked CD59 and weak CD55 expression were found in normal brains, but these complement regulators were virtually absent in ischaemic lesions. Modest amounts of mannose-binding lectin (MBL), MBL-associated serine protease-2 and factor B were found in both ischaemic lesions and controls. These data suggest that increased deposition of complement components combined with decreased expression of complement regulators is a possible mechanism of tissue damage during ischaemia in human brain.
Introduction Stroke is a serious neurological disease with a major impact on long-term disability and is one of the leading causes of death worldwide. Except for the thrombolytic therapy for a group of patients during a limited therapeutic time window, there is no effective treatment today that can improve a functional outcome in stroke. Even a short period of ischaemia can cause tissue damage, where a reperfusion is known to enhance and even widen the area of damage in the brain. It is, therefore, recognized that ischaemia-reperfusion injury (IRI) is a main mechanism of brain damage after stroke [1]. Limited data, however, are available about the mechanisms of tissue damage in human stroke, but it is known that the complement system can play a deleterious role in IRI [2]. Moreover, the complement system has been identified as a conductor of inflammation triggering further tissue damage in experimental ischaemia [3]. Thus, deposition of C1q [4–6] and C3 [7] in the brain has been detected in animal stroke models, and experimental treatment with the C9 component was shown to increase the volume of cerebral infarction in neonatal rats [8]. In contrast to animal studies, there are few data concerning the local complement activation in human ischaemic stroke [9].
The pathways responsible for complement-induced IRI may vary under different conditions and between different organs. Previous studies indicated the role of both the classical [10] and the mannose-binding lectin (MBL) complement pathways as possible triggers for tissue damage [11]. In addition, the alternative pathway appears to play a more crucial role in neuronal cell death than previously indicated [12]. The alternative pathway can be activated directly, but it is also important as a means of amplification of the initial classical pathway activation [13]. It has been shown that circulating natural IgM are trapped and that the complement is activated in tissues undergoing IRI [14]. Deposition of C-reactive protein (CRP) was found in human infarcted myocardium [15] and rat liver [16], whereas the binding of IgM to injured tissues was observed in murine skeletal muscle [17] and intestinum [18]. Serum IgM antibodies in patients with chronic sensorimotor polyneuropathy and IgM monoclonal gammopathy were shown to trigger demyelination of peripheral nerves due to complement activation [19]. Moreover, recent studies showed that reperfusion of ischaemic tissues elicits an acute inflammatory response involving the complement system which is activated by natural IgM. Therefore, a natural IgM-mediated innate autoimmunity response is more likely responsible for the detrimental consequences in ischaemic diseases [20].
2009 The Authors Journal compilation 2009 Blackwell Publishing Ltd. Scandinavian Journal of Immunology 69, 555–562
555
556 Complement in Human Brain Ischaemia E. D. Pedersen et al. .................................................................................................................................................................. Recently, the role of brain cells, especially microglial cells [21], and their implications in brain diseases in response to different pathological processes and body systems, has been a subject of debate. The impact of complement regulatory proteins in IRI and stroke is currently uncertain. It is known that CD55 is expressed by neurones in response to chronic inflammation in the central nervous system (CNS) associated with complement activation [22]. However, it has been speculated that regulatory proteins may also play a role in IRI [23]. Loss of regulatory proteins, e.g. CD59, has been shown to exacerbate IRI in mice [24] and promote neuronal cell lysis [25]. CD59 is shown to be lost from myocardial cells undergoing IRI [26], but the role of complement regulatory proteins in human stroke remains unclear. The aim of this study was, therefore, to evaluate possible local activation of the complement system during brain ischaemia and stroke in humans, with emphasis on deposition of individual components from the different pathways, as well as on expression of key complement regulatory proteins.
Materials and methods Patient demographics and tissue specimens. Ten patients with a clinical history of acute brain ischaemia or ischaemic stroke, who had died and were referred to the Department of Pathology for autopsy, were included in this study. The demographic data and pathological findings are listed in Table 1. The age of ischaemic lesions was estimated from the clinical history, CT findings and microscopical criteria. None of the patients had been treated with thrombolytic therapy - tissue plasminogen
activator (tPA). Autopsy was performed within 3 days after death. Cerebral tissue specimens were obtained from ischaemic lesions or infarctions and from remote sites of the same brain sections to account for general postmortem changes unrelated to the ischaemic lesions. In addition, brain tissue sections from the non-ischaemic brains of 10 individuals, matched for sex and age, and without previous evidence of acute ischaemia, ischaemic stroke or any inflammatory diseases, were used as negative controls. The study protocol was approved by the regional ethics committee. Permission to perform the autopsies was obtained from the patients’ relatives. Antibodies and reagents. The following primary antibodies were used: Rabbit polyclonal anti-C1q, anti-C3c and anti-C4d (Biomedica, Wien, Austria); mouse monoclonal anti-C5b-9 (aE11) (Diatec, Oslo, Norway); rabbit polyclonal anti-C9 (Wako Chemicals, Neuss, Germany); mouse monoclonal anti-CD35, anti-CD55 and antiCD59 ⁄ clone H19 (BD PharMingen, San Diego, CA, USA); mouse monoclonal anti-CD46 (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA); mouse monoclonal anti-CD59 ⁄ clone BRIC229 (National Blood Service, Bristol, UK); mouse monoclonal anti-factor B (Quidel, San Diego, CA, USA); mouse monoclonal antiMBL clone HYB 131-01, 131-10 and 131-11 (Antibodyshop, Gentofte, Denmark); mouse monoclonal anti-MBL clone 3F8 and IC10 (kindly provided by Prof. G. Stahl); rabbit polyclonal anti-MASP-2 [produced in one of the author’s (M.R.D.) laboratories]; rabbit polyclonal antiIgM (Lab Vision, Fremont, CA, USA); rabbit monoclonal anti-CRP (Abcam, Cambridge, UK); rabbit polyclonal anti-ITGAM (CD11b) (Atlas Antibodies AB, AlbaNova University Center, Stockholm, Sweden); mouse monoclo-
Table 1 Characteristics of the study population.
Case
Age
Sex
Underlying cause of death
1
55
m
Cerebral infarction
2
38
m
Aortic dissection
3 4 5
77 76 36
m f f
Cardiovascular disease Cardiovascular disease Oligodendroglioma
6
82
m
Cerebral infarction
7 8 9
85 66 64
f m f
10
77
m
Cardiovascular disease Cerebral infarction Post-operative complications with multi organ failure Lung cancer
Pathological findings
Brain sections
Age of lesion
Complement proteins
Other proteins
Thrombosis a.basilaris, vertebralis and carotis communis. Cerebral infarctions in cerebellum, pons, hippocampus, thalamus Massive cerebral infarction in the left hemisphere Massive hypoxic injury Hypoxic injury Cerebral infarction in the parietal brain region Thrombosis of the right a.cerebri media, anterior and carotis interna. Cerebral infarction in the right hemisphere Micro infarctions Massive cerebral infarction Massive hypoxic injury
Cerebral cortex Cerebellum
7 days 3 days
C1q, C4d, C3c, C9
CRP, IgM
Cerebral cortex
2 days
C1q, C4d, C3c, C9
Hippocampus Hippocampus Hippocampus Cerebral cortex Cerebral cortex
