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,§,¶,¶
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Department of Anatomy and Cell Biology, University of Marburg, Marburg, Germany;
Department of Microbiology and Immunology, University of Leicester, Leicester, United Kingdom;
Pharmacia & Upjohn SpA, Nerviano Mi, Italy;
§
Melacure Therapeutics AB, Uppsala, Sweden;
¶
Newron Pharmaceuticals Spa, Milan, Italy;
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Medical Research Council, Immunochemistry Unit, Department of Biochemistry, University of Oxford, Oxford, United Kingdom; and
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Institute for Medical Microbiology and Hygiene, Johannes Gutenberg-University of Mainz, Mainz, Germany
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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30 plasma proteins with an associated
group of cell membrane proteins. Activation of complement by the
classical, the alternative and the recently discovered lectin pathway
generates opsonins, inflammatory mediators, and cytolytic protein
complexes which play an essential role in clearing microorganisms and
tissue damage products (4). It has recently been suggested
that local activation of complement in the CNS is of pathophysiological
significance in both degenerative and inflammatory neurological
diseases including Alzheimer’s (2, 5, 6, 7, 8, 9) and Parkinson’s
dementia (10), supranuclear palsy (11),
Pick’s disease (12), multiple sclerosis (13, 14), cerebral malaria (15), meningoencephalitis
(16), scrapie (17, 18), and cerebrovascular
disorders (19). Increased biosynthesis of various
complement factors in the CNS has also been reported in experimental
animal models of neurodegeneration or neuroinflammation such as
peripheral or central axotomy (20, 21, 22, 23, 24), excitotoxic kainic
acid lesions (21, 25), exposure to neurotoxins
(26), and experimental allergic and virus-induced
encephalitis (27) and in cultured microglial cells
(28, 29, 30, 31, 32). By using a combination of techniques to monitor
and localize biosynthesis in vivo, we provide evidence that
experimentally induced cerebral ischemia causes a dramatic increase of
C1q in brain microglia resulting in markedly enhanced levels of C1q
functional activity in cerebrospinal fluid (CSF). |
Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Restriction enzymes, RNase A, and RNA polymerases were purchased from Boehringer Mannheim (Mannheim, Germany). [35S]CTP was obtained from New England Nuclear (Boston, MA). All chemicals, unless otherwise stated, were purchased from Sigma (Deisenhofen, Germany). X-ray films (Hyperfilm-ßmax) were purchased from Amersham-Pharmacia (Uppsala, Sweden). Photo emulsion, developer (full strength), and fixer (rapid fix) were from Kodak (Integra Biosciences, Fernwald, Germany). Wistar rats were from Charles River (Calco, Italy). Animal care and procedures were conducted according to institutional guidelines.
Induction of global ischemia and experimental groups
Transient forebrain ischemia was induced in 39 male Wistar rats (200–250 g) using the four vessel occlusion model according to the design of Pulsinelli and Brierley (33). Briefly, animals were anesthetized with chloral hydrate (350 mg/kg i.p.), and the vertebral arteries were electrocauterized in the alar foramina of the first cervical vertebra. The carotid arteries were isolated and transiently occluded with atraumatic clips for 15 min. Animals not showing isoelectric cortical electroencephalographic activity during ischemia and animals that did not fully lose their righting reflex after carotid artery occlusion were excluded from the study. Core body temperature was maintained at 37 ± 0.5°C during ischemia and for 2 h post ischemia with a feedback-regulated heating pad (LSI termist, Milan, Italy). In the sham animals, only the vertebral arteries were occluded. In addition, a group of untreated rats served as controls. Animals were sacrificed at 1, 4, 24, and 72 h after the ischemic insult. Their brains were randomly divided into two groups and either frozen in isopentane (-40°C) for in situ hybridization (n = 2–8) or processed for immunocytochemistry (n = 3–4).
In situ hybridization
For cellular localization of C1q mRNA, 35S-labeled sense and antisense RNA probes were generated by in vitro transcription from an internal 425-bp BamHI/PstI subfragment (cloned in pBluescript II KS-) of clone pRB-15, a cDNA transcript of mRNA encoding the B-chain of rat C1q (34). Labeling of the cRNA using [35S]CTP was performed according to the method of Melten et al. (35). With the use of T7 RNA polymerase, in vitro transcription yielded a cRNA in antisense orientation labeled with a specific activity of 430,000 Ci/mmol. Transcription with T3 RNA polymerase generated a sense strand probe labeled with a specific activity of 450,000 Ci/mmol. In situ hybridization histochemistry was performed according to the previously reported protocol (34, 36). Briefly, cryostat sections were fixed in 4% formaldehyde, 0.1 M PBS for 1 h at room temperature, after three washes in 0.05 M PBS, pH 7.4, for 10 min each. Slides were transferred to 0.1 M triethanolamine (TEA),4 pH 8.0, and incubated in TEA supplemented with acetic anhydride (0.25% v/v) for 10 min at room temperature, stirring rapidly. Sections were then rinsed in 2x SSC and dehydrated in ethanol (50–100%). Radioactive cRNA probes were diluted in hybridization buffer (50% formamide, 10% dextran sulfate, 3x SSC, 50 mM sodium phosphate (pH 7.4), 1x Denhardt’s (0.02% w/v Ficoll 400, polyvinylpyrrolidone 0.02% w/v, BSA 0.02% w/v), 0.1 mg/ml yeast RNA) to a final concentration of 5 x 104 dpm/µl. DTT was added to a final concentration of 10 mM. Hybridization mix (40 µl) was applied; the cryostat sections were then coverslipped and incubated in a humid chamber at 55°C for 16 h. The next day the coverslips were removed in 2x SSC. Sections were treated with RNase A (20 µg/ml) at 37°C for 60 min to remove single-stranded RNA molecules. Successive washes were done at room temperature in 2x, 1x, 0.5x, and 0.2x SSC for 10 min and in 0.2x SSC at 60°C for 1 h. The tissue was then dehydrated by dipping sequentially in 70%, 80%, and absolute ethanol. Hybridized sections were exposed to x-ray films (Hyperfilm-ßmax) for 96 h.
For microscopic analysis, slides were dipped in Kodak NTB2 nuclear emulsion and stored at 4°C. After 2–3 weeks of exposure, autoradiograms were developed for 2 min (Kodak D19 full strength) and fixed for 4 min. As controls, sections were incubated in parallel with C1q cRNA probes in sense strand orientation, or sections were pretreated with RNase before hybridization with the antisense probe.
For nonradioactive detection, the C1q riboprobe was labeled with digoxigenin-UTP according to the manufacturer’s protocol (Boehringer Mannheim). The digoxigenin-labeled C1q riboprobe was diluted in hybridization buffer to a final concentration of 0.5 ng/ml, and hybridization was performed with the same incubation protocol as for radioactive in situ hybridization. Hybridization signals were visualized with sheep anti-digoxigenin Fab fragments coupled to alkaline phosphatase according to the manufacturer’s protocol (Boehringer Mannheim).
Hybridized sections were analyzed in dark and bright field illumination and were photographed with the Olympus AX70 microscope (Olympus Optical, Hamburg, Germany).
Semiquantitative image analysis of hybridized sections
Analysis of autoradiograms was conducted using the MCID M4 image analysis system (Imaging Research, St. Catharine’s, ON, Canada). From each animal in each group, x-ray film autoradiograms of six 20-µm-thick interval sections were digitized under constant light and camera conditions. Calibrated densitometry of areas of the parietal cortex from similar Bregma levels was performed yielding measurements of integrated optical density (IOD; area x average optical density) as described previously (37). The optical density values were calculated after subtraction of the film background, which was defined by hybridization with the sense strand probes. A radioactive standard (ARC 146B, American Radiolabeled Chemicals, St. Louis, MO) was used for calibration. Data were expressed as IOD in nanocuries per gram.
Statistics
Intergroup differences in the C1q mRNA levels were evaluated using the Kruskal-Wallis one-way ANOVA followed by the Mann-Whitney U test. In all tests, p < 0.05 was considered significant.
Single enzymatic immunostaining of C1q
Deparaffinized serial sections (5–7 µm thick) of Bouin-Hollande (fixative solution containing 6% (w/v) picric acid, 2.5% cupric acetate, 3.7% formaldehyde, and 1% glacial acetic acid) fixed brains were incubated overnight with primary Abs at room temperature. Rat C1q was detected by using a polyclonal goat antiserum directed against mouse C1q (IgG fraction, diluted 1:400) as described previously (34). After washing in 50 mM PBS (pH 7.4), containing 1% w/v BSA, an anti-goat IgG biotinylated secondary antiserum (dilution, 1:200) (Dianova, Hamburg, Germany) was applied (45 min at 37°C). After repeated rinsing in PBS/BSA, sections were incubated with a streptavidin-HRP complex (Amersham, 1:200) for 2 h at 37°C. Staining was performed using H2O2 and 3',3-diaminobenzidine (Sigma) (12.5 mg/100 ml PBS) as a substrate and nickel enhancement by adding ammonium nickel sulfate (70 mg/100 ml, Fluka, Buchs, Switzerland). Immunostained sections were analyzed and photographed with the Olympus AX70 microscope. Specificity of the C1q antiserum was determined by preabsorption with purified rat and mouse C1q (50 µM) as well as by Western blotting and C1q ELISA (data not shown).
Confocal laser scanning double-immunofluorescence microscopy for C1q and markers for astrocytes, neurons, and microglial cells
Double-immunofluorescence detection of anti-C1q reactivity and reactivity for the different established markers (anti-glial fibrillary acidic protein (GFAP) for astrocytes, antineuronal nuclei (NeuN) for neurons, and isolectin B4 for microglia) was performed as follows. Sections were incubated overnight at room temperature with a mixture of the polyclonal goat anti-mouse C1q Ab (IgG fraction, diluted 1:40) and a polyclonal rabbit anti-GFAP antiserum (1:400, Dianova, Hamburg, Germany) or the mouse monoclonal anti-NeuN Ab MAB377 (dilution 1:20, Chemicon International, Temecula, CA), or biotin-labeled isolectin B4 (dilution 1:30; Sigma, St. Louis, MO). C1q immunoreactivity was visualized with indocarbocyanine-conjugated anti-goat IgG (Dianova), diluted 1:200, and applied for 45 min at 37°C, resulting in a red-orange fluorescence labeling. GFAP immunoreactivity (as a specific marker for astrocytes) and NeuN immunoreactivity (as a specific marker for neuronal cell bodies and nuclei) were visualized with biotinylated anti-rabbit IgG (Dianova) and biotinylated anti-mouse IgG (Dianova), respectively, both diluted 1:200. The secondary Abs were applied for 45 min at 37°C, followed by incubation with Alexis 488-conjugated streptavidin (MoBiTec, Göttingen, Germany) for 2 h at 37°C, resulting in a green fluorescence. Biotinylated isolectin B4 was visualized with Alexis 488-conjugated streptavidin for 2 h at 37°C, resulting in a green fluorescence. Sections were analyzed with the Olympus Fluoview confocal laser scanning microscope (Olympus Optical) and false color confocal images were printed with a digital color printer (Sony, Tokyo, Japan).
Measurement of C1q functional activity in the CSF by C1q-dependent hemolysis assay
In a separate set of experiments, rats (n = 3) for each of the different groups were implanted with an intrathecal catheter 1 week before the experiment to collect CSF samples. At different time points after global ischemia (1 h, 24 h and 72 h), 50-µl samples were drawn. C1q levels in these samples were determined by a sensitive C1q-dependent hemolysis assay. The assay was adjusted with serial dilutions of four samples to determine the appropriate sensitivity range. Thereafter, an aliquot (5 µl) of each CSF sample was assayed in duplicate, by adding the sample to 50 µl sheep erythrocytes (Tissue Culture Services, Botolph Claydon, U.K.) sensitized with rabbit anti-sheep erythrocyte Abs at a concentration of 2 x 108 cells/ml. Both the sample and the EA cells were incubated at 37°C for 30 min, then 50 µl of a 1:50 dilution of C1q-deficient human serum (Sigma, Deisenhofen, Germany) was added, and the incubation was continued for 1 h at 37°C. The remaining unlysed cells were then spun down, and the optical density (OD412) of the supernatant was read and expressed as percentage lysis. The assay buffer was dextrose-gelatin-Veronal buffer with 0.15 mM CaCl2 and 0.5 mM MgCl2. The titer of C1q activity was expressed in arbitrary units relative to a human C1q standard. It was not considered appropriate to express C1q in absolute units (e.g., micrograms per milliliter), because the relative activities of rat and human C1q in the assay were not determined.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Biosynthesis of C1q was examined at the mRNA level in rat brain
sections at different time points after global transient ischemia and
compared with sham-operated and control rats, respectively, which is
shown on low magnification x-ray micrographs in Fig. 1
. In situ hybridization demonstrated a
slight increase of C1q mRNA levels throughout the brain within the
first hour (Fig. 1, B and C) after ischemia, with
a more pronounced elevation after 24 h (Fig. 1D). A
dramatic increase of C1q mRNA levels was observed 72 h
postischemia (p.i.), especially in the hippocampus, in ventroposterior
thalamic nuclei, and in the parietal neocortex (Fig. 1E). At
low magnification, it was already obvious that C1q mRNA expression was
absent from neuronal layers of cerebral cortex and hippocampus. High
power bright field analysis of emulsion-dipped slides (data not shown)
consolidated the observation that C1q mRNA was absent from neurons in
both control and all experimental groups and exclusively present in
small cells distributed throughout gray and white matter which were
identified as microglial cells (see below).
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). Control and
sham-operated animals displayed only low levels of C1q mRNA (3.80 and
5.06 nCi/g tissue, respectively). At 1 and 4 h p.i., C1q mRNA
expression was significantly increased >3-fold over control values
(16.60 and 18.20 nCi/g tissue, respectively). After 24 h, a 70- to
100-fold increase of C1q mRNA levels (375.48 nCi/g) was observed as
compared with control or sham. At 72 h p.i., levels of C1q mRNA
(658.10 nCi/g) were in excess of 100- and 200-fold as compared with
control and sham, respectively. The intensities of hybridization
signals in the sham control at 24 and at 72 h were identical (data
not shown).
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Both low and high magnification immunocytochemistry and in situ
hybridization demonstrate that microglial cells, but no other resident
cells in the brain, are the source of constitutive biosynthesis and of
ischemia-induced increase in C1q biosynthesis throughout the rat brain.
This is shown, for example, in the hippocampal region 24 h after
ischemia (Fig. 2).
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). Isolectin B4, in addition, also
stains endothelial cells (Fig. 3I). The endothelial cells
stained for isolectin B4 are negative for C1q (Fig. 3H). C1q
immunoreactivity was completely absent in neuronal cell bodies (Fig. 3, D–F) identified by using mouse anti neuronal nuclei Ab
NeuN, an established marker for neurons (Fig. 3F).
Furthermore, no expression of C1q in GFAP-positive astrocytes was
observed (Fig. 3, A–C). The data shown in Fig. 3 are from
sections through the parietal cortex 24 h after ischemia. These
patterns of expression were observed throughout all areas of the brain
at any of the time points after the ischemic insult.
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Analysis of C1q functional activity in CSF
To quantify secretion of functionally active C1q into CSF, samples
were collected at different time points after the ischemic insult, and
functional activity of C1q was determined in a C1q-dependent hemolytic
assay. Samples were assessed for serum leakage by measuring
OD412 as an indicator of hemoglobin
contamination, and CSF samples with serum contamination were excluded.
At 24 h p.i., the level of hemolytically active C1q was markedly
elevated (>5-fold compared with control and >3-fold compared with
sham; see Fig. 4). At 72 h p.i., the
levels of C1q functional activity in CSF were still more than twice as
high as in sham animals. The relative lower level of functionally
active C1q at 72 h compared with 24 h may be explained by C1q
consumption (i.e., removal from the fluid phase due to classical
pathway activation or binding of C1q to its receptors)
(1).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The sustained rise of C1q mRNA levels and biosynthesis in microglial cells in response to global ischemia identifies this complement component as a sensitive and specific marker of microglial activation. This is in line with our previously published observations that the level of C1q biosynthesis monitored by Northern blot, in situ hybridization, and immunocytochemistry 1) is low and is restricted to microglial cells in normal rat and mouse brain and 2) is strongly up-regulated in CNS microglia after viral infection and experimental allergic encephalitis (27, 34, 38). We found that constitutive and lesion-induced C1q biosynthesis was absent in any brain-resident cells other than cells of the microglia/macrophage lineage. This conforms to other reports of experimental brain lesions in vivo (22, 39) and to in vitro data (28, 29) providing strong evidence for microglial biosynthesis of C1q.
These findings, however, are in contrast to recently published immunocytochemical data which were interpreted to indicate that C1q biosynthesis is absent in normal mouse brain and induced de novo and exclusively in neurons during reperfusion after transient focal cerebral ischemia in the mouse brain (40). Thus far, this conclusion has not been corroborated by in situ hybridization analysis. Thus, it cannot be ruled out that the immunostaining for C1q in neurons is a result of C1q deposition on neurons in the infarct area rather than due to neuronal C1q biosynthesis. In fact, there is evidence that in the course of cerebrovascular disease and Alzheimer’s disease, serum proteins like Ig, fibrinogen, and complement factors are deposited in the brain parenchyma (41). Although there is accumulating evidence for enhanced cerebral biosynthesis of C1q in Alzheimer’s disease brain, based on Northern blot, RT-PCR, and Western blot results, the issue of neuronal vs nonneuronal biosynthesis of C1q in normal human brains and in Alzheimer’s disease brains is still controversial (2, 5, 7, 8, 41, 42). Likewise, in experimental studies of different types of inflammatory and neurodegenerative brain lesions, the neuronal vs nonneuronal biosynthesis of C1q is a matter of controversy (21, 22, 24, 25, 26, 27).
It has been postulated that cerebral complement and complement receptor activation is causal for neuronal damage, both in encephalitis and in neurodegeneration (3, 9), suggesting that this is also the case in cerebral ischemia. The mechanisms by which locally produced complement factors may act cytotoxically on activation include the formation of the membrane attack complex, triggering phagocytosis and free radical release from complement receptor-bearing cells and synergistic interactions with proinflammatory cytokines (3).
In this context, it remains to be clarified whether and how an increase in the local production of C1q in CNS tissue may contribute to the pathology of CNS disease. It is conceivable that raised levels of C1q may promote inflammation in two ways: 1) they may lead to increased levels of functionally active C1 complexes, thus driving a local activation of the classical complement activation cascade (1); 2) they might trigger cellular responses by binding to C1q receptors (1, 43). The potential benefits of using complement inhibitors as a novel therapeutic approach in the treatment of inflammatory and degenerative neurological diseases is highlighted by a recent report of Huang et al. (40) which demonstrates a reduced cerebral infarct volume and neuronal protection by infusion of a hybrid molecule that simultaneously inhibits complement activation and selectin-mediated adhesion in a murine experimental model of focal cerebral ischemia in mice. These results, however, do not permit assessment of the contribution of enhanced C1q biosynthesis, because the complement-inhibitory activity of the hybrid molecule used in this study becomes effective further downstream of C1 activation only and may inhibit each of the three activation routes of complement. The contribution of CNS biosynthesis of C1q under pathological conditions can be evaluated only in either C1q-deficient mouse strains (43) or using specific inhibitors of C1 activation (44).
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Acknowledgments |
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Footnotes |
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2 Data were presented in part at the 26th Annual Meeting of the Society for Neuroscience, Washington, DC, 1996 (45 ), and the Fourth International Workshop on C1 and Collectins, Mainz, Germany, October 3–5, 1997 (46 ).
3 Address correspondence and reprint requests to Dr. Eberhard Weihe, Department of Molecular Neuroimmunology, Institute of Anatomy and Cell Biology, Philipps-University of Marburg, Robert Koch Strasse 6, 35033 Marburg, Germany.
4 Abbreviations used in this paper: TEA, triethanolamine; CSF, cerebrospinal fluid; NeuN, antineuronal nuclei; p.i., postischemia.
Received for publication September 22, 1999. Accepted for publication March 6, 2000.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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