Inhibition of the alternative complement activation pathway in traumatic brain injury by a monoclonal anti-factor B antibody: a randomized placebo-controlled study in mice
© Leinhase et al; licensee BioMed Central Ltd. 2007
Received: 19 March 2007
Accepted: 02 May 2007
Published: 02 May 2007
The posttraumatic response to traumatic brain injury (TBI) is characterized, in part, by activation of the innate immune response, including the complement system. We have recently shown that mice devoid of a functional alternative pathway of complement activation (factor B-/- mice) are protected from complement-mediated neuroinflammation and neuropathology after TBI. In the present study, we extrapolated this knowledge from studies in genetically engineered mice to a pharmacological approach using a monoclonal anti-factor B antibody. This neutralizing antibody represents a specific and potent inhibitor of the alternative complement pathway in mice.
A focal trauma was applied to the left hemisphere of C57BL/6 mice (n = 89) using a standardized electric weight-drop model. Animals were randomly assigned to two treatment groups: (1) Systemic injection of 1 mg monoclonal anti-factor B antibody (mAb 1379) in 400 μl phosphate-buffered saline (PBS) at 1 hour and 24 hours after trauma; (2) Systemic injection of vehicle only (400 μl PBS), as placebo control, at identical time-points after trauma. Sham-operated and untreated mice served as additional negative controls. Evaluation of neurological scores and analysis of brain tissue specimens and serum samples was performed at defined time-points for up to 1 week. Complement activation in serum was assessed by zymosan assay and by murine C5a ELISA. Brain samples were analyzed by immunohistochemistry, terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) histochemistry, and real-time RT-PCR.
The mAb 1379 leads to a significant inhibition of alternative pathway complement activity and to significantly attenuated C5a levels in serum, as compared to head-injured placebo-treated control mice. TBI induced histomorphological signs of neuroinflammation and neuronal apoptosis in the injured brain hemisphere of placebo-treated control mice for up to 7 days. In contrast, the systemic administration of an inhibitory anti-factor B antibody led to a substantial attenuation of cerebral tissue damage and neuronal cell death. In addition, the posttraumatic administration of the mAb 1379 induced a neuroprotective pattern of intracerebral gene expression.
Inhibition of the alternative complement pathway by posttraumatic administration of a neutralizing anti-factor B antibody appears to represent a new promising avenue for pharmacological attenuation of the complement-mediated neuroinflammatory response after head injury.
All experiments were performed in adult male mice of the C57BL/6 strain (n = 89 in total) purchased from Jackson Laboratory (Bar Harbor, ME). The mice were bred in a selective pathogen-free (SPF) environment and under standardized conditions of temperature (21°C), humidity (60%), light and dark cycles (12:12 h), with food and water provided ad libitum. Experiments were performed in compliance with the standards of the Federation of European Laboratory Animal Science Association (FELASA) and were approved by the institutional animal care committee (Landesamt für Arbeitsschutz, Gesundheitsschutz und technische Sicherheit Berlin, Berlin, Germany, No. G0099/03).
Mice were subjected to experimental TBI using a standardized weight-drop device, as previously described [26, 35, 39, 40]. In brief, after induction of isoflurane anesthesia, the skull was exposed by a midline longitudinal scalp incision. The head was fixed and a 250 g weight was dropped on the skull from a height of 2 cm, resulting in a focal blunt injury to the left hemisphere. After trauma, the mice received supporting oxygenation with 100% O2 until fully awake. The extent of posttraumatic neurological impairment was assessed at defined time intervals after trauma (t = 1 h, 4 h, 24 h, and 7 days) using a standardized Neurological Severity Score (NSS), as described below.
The inhibitory monoclonal anti-factor B antibody (mAb 1379) used in this study was previously described and the selected dosage was in the titrated range used in other studies on murine models of inflammation [34, 36, 37]. The antibody itself does not have any complement-activating properties. Mice were randomly assigned to two treatment groups: (1) Systemic injection of 1 mg mAb 1379 in 400 μl phosphate-buffered saline (PBS) at 1 hour and 24 hours after trauma; (2) Systemic injection of vehicle only (400 μl PBS), as placebo control, at identical time-points after trauma. Concealed allocation to the two treatment cohorts was performed after assessment of the baseline NSS at 1 hour after trauma, in order to ensure equal injury severity between the groups. The systemic (i.p.) route of administration and the time window of injection were selected based on the breakdown of the blood-brain barrier (BBB) for up to 24 hours after trauma [38, 41]. This allows a "time window" for peripherally administered compounds to reach the intrathecal compartment and exert pharmacological effects in the CNS [26, 39, 40, 42]. Furthermore, the systemic injection early after trauma represents an approach with potential clinical implications. In order to induce a continuing complement inhibition during the acute inflammatory phase in the first days, injections were repeated at 24 hours.
Subgroups of mice (n = 10 per group and time-point) were euthanized by isoflurane anesthesia and decapitated at t = 4 h, 24 h, and 7 days. Brains were immediately extracted, snap-frozen in liquid nitrogen and stored at -80°C until analysis by immunohistochemistry, TUNEL histochemistry and real-time RT-PCR. In addition, serum samples were collected at identical time-points for determination of complement activation levels. Sham-operated and untreated normal mice served as negative controls.
Neurological Severity Score (NSS)
A previously characterized 10-parameter score was used for assessment of posttraumatic neurological impairment, as described elsewhere in detail [41, 43]. The NSS was assessed in a blinded fashion by two different investigators at the time-points t = 1 h, 4 h, 24 h, and 7 days after trauma. The score comprises 10 individual parameters, including tasks on motor function, alertness, and physiological behavior, whereby one point is given for failure of the task, and no point for succeeding. A maximum NSS score of 10 points indicates severe neurological dysfunction, with failure of all tasks.
Mouse C5a ELISA
Serum levels of the complement anaphylatoxin C5a were determined by a mouse-specific ELISA developed in the laboratory of Dr. P.A. Ward (Ann Arbor, MI), as previously described [35, 44]. In brief, ELISA plates (Immulon 4HBX, Thermo Labsystems, Milford, MA) were coated with 5 μg/ml of purified monoclonal anti-mouse C5a IgG (BD Pharmingen, San Diego, CA). After blocking of non-specific binding sites with 1% milk (Roth, Karlsruhe, Germany) in PBS (Gibco-Invitrogen, Carlsbad, CA) containing 0.05% TWEEN 20 (Sigma-Aldrich), the plate was coated with 100 μl of each serum diluted 1:20 (in 0.1% milk in PBS containing 0.05% TWEEN) and murine recombinant mouse C5a at defined concentrations for establishing the standard curve. After incubation and subsequent washing steps, biotinylated monoclonal anti-mouse C5a antibody was added at 500 ng/ml (BD Pharmingen) followed by washing steps and incubation with streptavidin-peroxidase at 400 ng/ml (Sigma-Aldrich).
For colorimetric reaction, 0.4 mg/ml o- phenylenediamine dihydrochloride with 0.4 mg/ml urea hydrogen peroxide in 0.05 M phosphate citrate buffer (Sigma-Aldrich) was added and the color reaction was stopped with 3 M sulfuric acid. Absorbance was read at 490 nm using a "SpectraMax 190" reader (Molecular Devices, Sunnyvale, CA). All samples were analyzed in duplicate and results were calculated from the means of duplicate sample analysis. The standard curve was linear from 0.1 ng/ml to 50 ng/ml.
Quantification of alternative pathway complement activity
Immunohistochemical stainings of serial coronal cryosections (8 μm) of brain tissue were performed using a biotin/avidin/peroxidase technique with diaminobenzidine tetrahydrochloride as chromogen (Vector, Burlingame, CA). The following primary antibodies were used as cell-markers: monoclonal anti-NeuN for neurons (1:2,000; Chemicon, Hampshire, UK); polyclonal rabbit anti-GFAP for astrocytes (1:100; Shandon Immunon, Pittsburgh, PA) and monoclonal rat anti-CD11b for microglia and monocytes/macrophages (1:100; Accurate Chemical, Westbury, NY). For negative control, non-immunized IgG (Vector) was used at equal dilutions.
The terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) technique was applied to determine the extent of neuronal cell death in tissue sections. Herefore, the commercially available ''Fluorescein In Situ Cell Death Detection Kit'' (Roche Diagnostics GmbH, Mannheim, Germany) was used according to the manufacturer's instructions, as previously described . In brief, slides were dried for 30 min followed by fixation in 10% formalin solution at RT. After washing in PBS, sections were incubated in ice-cold ethanol-acetic acid solution (3:1), washed in PBS and incubated with 3% Triton X-100 solution for 60 min at RT for permeabilization. Slides were then incubated with the TdT-enzyme in reaction buffer containing fluorescein-dUTP for 90 min at 37°C. Negative control was performed using only the reaction buffer without TdT enzyme. Positive controls were performed by digesting with 500 U/ml DNase grade I solution (Roche). To preserve cells for comparison, slices were covered with Vectashield® mounting medium containing 4',6'-diamino-2-phenylindole (DAPI; Vector). All samples were evaluated immediately after staining using an ''Axioskop 40'' fluorescence microscope (Zeiss, Germany) at 460 nm for DAPI and 520 nm for TUNEL fluorescence. Data were analyzed by Alpha digi doc 1201 software (Alpha Innotech, San Leandro, CA).
Murine primer sequences used for real-time RT-PCR analysis of intracerebral gene expression
Gene ID at NCBI *
GeneBank Accession No.
Length of amplicons
Order No. Qiagen
commercially available Genexpression Assay QuantiTect Mm_GAPD
commercially available Genexpression Assay QuantiTect Mm_Bcl-2
commercially available Genexpression Assay QuantiTect Mm_ Tnfsf6
Statistical analysis was performed using commercially available software (SPSS 9.0 for Windows™). Differences in serum complement activity levels and in intracerebral gene expression levels between the groups were determined by the unpaired Student's t-test. The repeated measures analysis of variance (ANOVA) was used for assessing differences in neurological scores (NSS). A P-value < 0.05 was considered statistically significant.
mAb 1379 inhibits complement activation after TBI
The assessment of intracerebral cell death by TUNEL histochemistry revealed a dramatic increase in TUNEL-positive neurons in the injured left hemispheres of PBS-injected mice at 4 hours after trauma, as previously described for this TBI model . TUNEL-positive cells were detected within the contused area (Fig. 6L) and the hippocampus (not shown) of the injured hemisphere for for up to 7 days after trauma, as compared to sham-operated animals (Fig. 6K). In contrast, the mAb 1379 treated mice showed a clearly attenuated extent of intracerebral cell death in the ipsilateral hippocampus (not shown) and cortex around the contusion zone for up to 7 days after trauma (Fig. 6M). Immunohistochemical staining of adjacent sections to those analyzed by TUNEL histochemistry by cell markers for neurons (anti-NeuN), astrocytes (anti-GFAP), and microglia and infiltrating leukocytes (anti-CD11b), revealed that neurons were the predominant TUNEL-positive cell type in all sections taken from the injured hemisphere in PBS-treated mice. Neurons were also confirmed as the predominant TUNEL-positive cell-type by their typical cellular size, morphology, and position in typical neuronal layers. In addition, some infiltrating leukocytes within the contusion site were shown to be TUNEL-positive at the time-point of 7 days after trauma (Fig. 6L). TUNEL-positive cells and the extent of cortical tissue destruction were less apparent in the contralateral (right) hemisphere as compared to the injured (left) hemisphere at all time-points assessed after trauma (data not shown). The representative microphotographs shown in Fig. 6 were highly reproducible in all tissue sections and animals assessed.
Intracerebral gene regulation
After head trauma, the mAb1379-injected mice showed a significant upregulation of the protective Bcl-2 and C1-Inh genes for up to 7 days, as compared to placebo-injected or sham-operated mice (P < 0.05, unpaired Student's t-test; n = 6 per gene, time-point, and TBI group Fig. 7). In contrast, Fas gene expression in injured brains showed different kinetics of regulation, with mRNA levels being significantly elevated in both TBI groups (placebo and mAB 1379) as early as 4 hours after trauma, compared to sham-operated mice (P < 0.05, unpaired Student's t-test; n = 6 per gene, time-point, and TBI group Fig. 7). As opposed to the Bcl-2 and C1-Inh genes, no significant differences in Fas gene expression were seen between the mAB 1379 and placebo-control groups at 4 hours and 7 days after trauma (Fig. 7). However, at 24 hours, Fas mRNA levels were significantly suppressed in the treatment group, reaching similar low levels as the sham controls (P < 0.05, mAb1379 vs. PBS group; Fig. 7).
Therapeutic modalities for inhibition of the complement cascade have been assessed in different models of brain injury in the past [5, 17, 23]. Most of these studies have used pharmacological approaches which led to complete "shut-down" of the complement system at the level where the three different activation pathways merge by inhibiting the C3 convertases (Fig. 1) [20, 21, 25, 26]. A recent experimental study from our laboratory suggests, however, that the alternative pathway may be of particular importance in mediating neuroinflammation and neuronal cell death after head injury, based on studies in factor B gene knockout (fB-/-) mice . The selective inhibition of the alternative pathway only has received increasing attention in various inflammatory diseases outside the CNS, due to recent findings which support its essential role in contributing to secondary tissue injury [31, 32]. Based on our recent findings of a significant neuroprotection in fB-/- mice after TBI, we sought to extrapolate these findings to a pharmacological model by targeted inhibition of the alternative pathway . We therefore used a newly available, highly specific and potent inhibitor of the alternative complement pathway, the mAb 1379 monoclonal anti-factor B antibody, in the identical head injury model. This antibody was previously shown to protect form inflammation and severity of disease in allergic airway inflammation, renal ischemia/reperfusion syndrome, and anti-phospholipid antibody-induced pregnancy loss in mice [34, 36, 37].
In the present study, we randomized adult male C57BL/6 to receive a systemic injection of either 1 mg mAb 1379 or placebo (vehicle only) at 1 hour and 24 hours after closed head injury. The selected dosage was in the titrated range used in previous studies on other murine models of inflammation [34, 36, 37]. The systemic (i.p.) route of administration and the time window of injection were selected based on the rationale that in this model system, the blood-brain barrier is breached as early as 1 hour after trauma, peaking at 4 hours, and persisting for up to 24 hours [38, 41]. These kinetics of blood-brain barrier opening offer a "time window of opportunity" for peripherally administered compounds to reach the intrathecal compartment and exert pharmacological effects in the inflamed CNS, as previously shown for other pharmacological agents [26, 39, 40, 42]. Furthermore, the post-trauma systemic injection within 1 to 24 hours after injury represents an approach with potential clinical implications [10, 14].
Our data demonstrate that the mAb 1379 represents a potent complement inhibitor after TBI, based on a significant attenuation of alternative pathway complement activity (zymosan assay) and a significant inhibition of complement anaphylatoxin C5a levels (ELISA data) at 4 and 24 hours after trauma, compared to placebo controls. However, while the injection of 1 mg mAb 1379 induced a complement inhibition for up to 24 hours, the repeated injection at this time-point was obviously not sufficient for sustaining a prolonged inhibition of complement activation until 7 days after injury. In other experimental models of inflammation, we have recently found that the hepatic factor B synthesis is increased due to initiation of the acute-phase response, thus necessitating higher doses of mAb 1379 for complete inhibition (Holers VM, Thurman JM; unpublished observations).
Aside from the shortcoming of limited complement inhibition related to the half-life of the compound, compensatory inflammatory reactions may also account for the lack of neurological improvement. These compensatory effects include the release of pro-inflammatory cytokines in the injured brain, such as tumor necrosis factor (TNF) and of interleukins (IL) -1β, -8, -12, -18, and other mediators of neuroinflammation [2, 45, 46]. Finally, the neurological score used in the present study (NSS), albeit widely used with success in previous studies on this model system [26, 27, 39–43], may not be sensitive enough to detect subtle changes in performance attributed to morphological alterations of cerebral tissue damage. Thus, other neurological testing systems may have to be applied in future studies to test the relevance of this compound in neurotrauma in more detail, including the Morris water maze for assessment of memory tasks.
Despite the lack of neurological improvement in the mAb 1379-treated mice, we observed an impressive extent of neuroprotection at the tissue level and a significant induction of neuroprotective genes in the injured brain. Specifically, the mAb 1379-treated mice had an attenuated extent of neuronal cell death and a preserved cortical microarchitecture for up to 7 days after head injury, compared to placebo controls. These promising findings imply that with a modified protocol of mAb 1379 administration, e.g. by higher doses or repeated injections every 24 hours for the first week, may lead to an increased extent of cerebral neuroprotection which will likely influence the outcome at a clinical-neurological level. Another strategy could involve the use of therapies targeted to the brain using CR2-linked chimeras which might provide more complete local control of complement activation [47, 48]. This hypothesis will have to be tested in future experimental studies.
The alternative pathway of complement activation appears to play a more crucial role in the pathophysiology of complement-mediated neuroinflammation after TBI than previously appreciated. In the present study, we extrapolated previous findings of neuroprotection in factor B gene-deficient (fB-/-) mice  to a pharmacological approach using a specific and potent inhibitor of the alternative complement pathway (mAb 1379). The randomized treatment protocol used in this experimental study on closed head injury in mice revealed the following mAb 1379-mediated beneficial effects, as compared to placebo controls:
(1) A significant attenuation of complement pathway activity at the level of the alternative pathway (zymosan assay) and overall at the level of anaphylatoxin formation (C5a ELISA).
(2) An impressive reduction of neuronal cell death (TUNEL) and a restoration of cortical cell layers in the injured hemisphere (immunohistochemistry).
(3) A significant upregulation of candidate neuroprotective genes in the injured hemisphere (real-time RT-PCR).
However, these neuroprotective effects at the tissue level did not extend to an improved neurological outcome or to reduced mortality in mAb 1379-treated mice, as compared to placebo controls. The observation of elevated factor B levels in the intrathecal compartment of severely head-injured patients further supports the pharmacological concept of a specific inhibition of factor B . However, prior to extrapolation to the clinical setting, further animal studies will be required for determining the optimal dosage and injection intervals in experimental models of head injury.
Central nervous system
enzyme-linked immunosorbent assay
glial fibrillary acidic protein
neuron-specific nuclear protein
- real-time RT-PCR:
real-time reverse transcriptase polymerase chain reaction
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
traumatic brain injury
terminal deoxynucleotidyl transferase
terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling.
Dr. Allison Williams is gratefully acknowledged for help with statistical analysis of the data. We furthermore thank Claudia Conrad, Malte Pietzcker and Carlo Farah for excellent technical assistance. This project was previously presented in part at the 123rdAnnual Congress of the German Society for Surgery (DGC), May 2–5, 2006, in Berlin, Germany, and at the XXI. International Complement Workshop, October 20–27, 2006, in Beijing, China. Parts of this work have been published in abstract form in the proceedings of these scientific meetings. This study was supported by the German Research Foundation (DFG) grants No. STA 635/1-1, STA 635/1-2 (to PFS), and STA 635/2-1, STA 635/2-2 (to PFS, OIS, WE); NIH grants R01 AI31105 (to VMH) and K08 DK64790 (to JMT); grants GM 61656 and GM 029507 (to PAW).
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