Background
Although the brain is considered to be an immune-privileged organ [
1,
2], devastating effects can occur in the brain during local inflammation, particularly in response to cerebral bacterial infection. The recruitment of immune cells from the circulation is critical for many life-threatening central nervous system (CNS) inflammatory diseases, such as bacterial meningitis [
3], multiple sclerosis [
4], and stroke [
5]. Neutrophils mobilized from the circulation in response to signals from the CXC family of chemokines, such as keratinocyte-derived chemokine (KC, CXCL1), and macrophage inflammatory protein-2 (MIP-2, CXCL2), are considered to be the first line of defense against bacteria [
6‐
9]. Once activated through cytokines, such as tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ), endothelial cells upregulate adhesion molecule expression, which enhances their interactions with the leukocytes and increases their transmigration into the brain [
10,
11]. Neutrophils have been identified as the key players in CNS inflammation, and strategies that block neutrophil recruitment have been demonstrated as beneficial for the treatment of many types of CNS inflammation [
12‐
14]. In a previous study, we showed that microglia play a dominant role in the innate immune response to infectious agents in the CNS [
15]. Additionally, it has been widely accepted that inflammatory cytokines, such as TNF-α and chemokines from glial cells, activate the endothelium, thereby increasing adhesion molecule expression and leukocyte recruitment [
16‐
18].
The complement system consists of approximately 40 soluble and membrane-bound proteins that play a central role in host defense against pathogens and inflammation initiation [
19,
20]. Although the liver is the primary source of complement production, increasing evidence from recent studies has shown that many types of resident cells in the CNS produce complement components [
21]. The expression of complement receptors, such as the C3a and C5a receptors on glial cells and neurons in the CNS, has been reported [
22,
23]. Further studies in animal models have provided evidence for the involvement of the complement system in modulating CNS inflammation [
24,
25]. In particular, systemic complement depletion reduces perihematomal brain edema and TNF-α production following experimental intracerebral hemorrhage [
26]. Moreover, C3
−/− mice exhibited less brain edema and less microglial activation and neutrophil infiltration around the clot after intracerebral hemorrhage [
27]. In a mouse model of meningitis induced by
Streptococcus pneumonia infection, C1q and C3 deficiency led to reduced cerebrospinal fluid (CSF) leukocyte counts in comparison with infected wild-type (WT) mice [
28]. Additionally, mice lacking C3 and its receptors developed increased burdens of West Nile virus in the CNS [
29]. C5a receptor-deficient mice with pneumococcal meningitis also showed lower CSF leukocyte counts and alleviated brain damage compared with WT mice. Moreover, treatment with C5-specific monoclonal antibodies prevented animal death in WT mice with pneumococcal meningitis [
30]. These studies suggest that the complement system plays a significant role in the CNS inflammatory response. However, the mechanisms underlying the contribution of complement components to immune cell recruitment into the brain parenchyma through the blood-brain barrier (BBB) remain unclear.
In this study, we systemically examined the detailed mechanisms underlying the contribution of complement components to leukocyte recruitment in response to cerebral lipopolysaccharide (LPS) administration. We first detected significantly reduced infiltrating neutrophils in the brain of C3-deficient (C3−/−) and C3a receptor-deficient (C3aR−/−) but not C6-deficient (C6−/−) mice. C3 deficiency also significantly reduced endothelial activation and leukocyte-endothelial interactions in brain postcapillary venules. Permeability changes in the BBB were comparable between WT and C3−/− mice. Adhesion molecules, such as E-selectin and vascular cell adhesion molecule 1 (VCAM-1), showed reduced expression levels in the brains of C3−/− mice in response to LPS administration, suggesting that C3 contributes to the activation of brain endothelium. Furthermore, TNF-α was not able to induce leukocyte recruitment in C3−/− mice. C3a directly stimulated cerebral endothelial activation in vitro. Taken together, these results demonstrate that C3a plays a critical role in endothelial activation and subsequent leukocyte recruitment in the brain in response to intracerebroventricular LPS administration.
Methods
Animals
Adult male wild-type (WT) C57BL/6J and BALB/c mice 7 to 8 weeks old weighing 20 to 25 g were obtained from the Model Animal Research Center, Nanjing University. C3-deficient (C3−/−) mice (C57BL/6J mice background) and C3a receptor-deficient (C3aR−/−) mice (BALB/c mice background) were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The C5aR−/− mice (C57BL/6J background) were obtained from Dr. Hu Weiguo (Shanghai Cancer Center and Institute of Biomedical Science, Shanghai Medical College, Fudan University, China). The C6-deficient (C6−/−) mice (C57BL/6J mice background) were a gift from Dr. Tod Merkel (Center for Biologics Evaluation and Research, Food and Drug Administration, USA). The animals were housed under a 12-h light/dark cycle under specific pathogen-free conditions with free access to food and water. All animal protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Nanjing Medical University. All animal experiments were carried out in a blinded and randomized fashion.
Intracerebroventricular LPS injection
The mice were anesthetized using an intraperitoneal (i.p.) injection of 200 mg/kg of ketamine and 10 mg/kg of xylazine. Subsequently, the mice were placed onto a rodent stereotaxic frame (David Kopf Instruments, Tujunga, CA, USA). The scalp was shaved, and a burr hole was drilled 1 mm caudal to the bregma and 2.0 mm lateral to the midline [
15,
31]. A total of 2 μL of 2 μg of LPS (
Escherichia coli serotype 0111:B4 strain; InvivoGen) or 0.2 μg of TNF-α (R&D systems, Minneapolis, MN, USA) was administered by intracerebroventricular (i.c.v.) injection using a 10-μl Hamilton microsyringe over a 3-min period. Control animals received an isovolumetric i.c.v. injection of saline. Body temperature was monitored using a rectal probe, and the mice were maintained under deep anesthesia at 36 ± 1 °C using a thermostatic heating system (Harvard Apparatus, MA, USA) throughout the experiment.
Immunohistochemical procedure
Mice under deep anesthesia and received i.c.v. LPS or saline injection were perfused through the heart with ice-cold 4 % formalin. The cerebral tissues were then removed and fixed in 4 % formalin for 48 h. Thick coronal sections were obtained at −1.0 to −3.0 mm from the bregma. Formalin-fixed tissues were embedded in paraffin and subsequently sectioned at a 4-μm thickness using a cryostat. Infiltrating neutrophils were detected using a Naphthol AS-D Chloroacetate-Specific Esterase kit (Sigma-Aldrich, St. Louis, MO, USA).
Intravital microscopy
The animals were anesthetized and monitored as described previously [
15]. A craniotomy was performed using a high-speed drill in the right parietal bone. Stripping the dura from the site exposed the brain pial vessels. Subsequently, the animals were intravenously (i.v.) administered rhodamine 6G (Sigma-Aldrich) (0.5 mg/kg, body weight) to label leukocytes. Leukocyte-endothelial interactions were recorded using an sCMOS camera (ORCA-Flash 4.0, HAMAMATSU) mounted onto a Nikon FN1 microscope. Three different postcapillary venules with diameters between 30 and 70 μm were chosen for observation. All experiments were recorded for subsequent playback analysis. Rolling leukocytes were defined as cells moving at a velocity less than that of erythrocytes. Cells remaining stationary for at least 30 s were considered adherent.
Blood-brain barrier permeability determination
The levels of albumin, a plasma protein that is normally excluded from the brain by the intact BBB, were used as an indicator for its integrity. The mice were anesthetized and perfused with 20 ml of ice-cold phosphate-buffered saline (PBS) to remove contaminated albumin from the circulation. Then, the concentration of albumin in brain homogenates was measured by Western blotting as previously described [
32].
Complement depletion
Four hours prior to i.c.v. LPS injection, hypocomplementemia was induced through the i.p. injection of cobra venom factor (CVF) (Biogen Sci & Tech Co., Kunming, China) in PBS into C57BL/6J mice at a dose of 10 μg/mouse (15 U/mouse). This treatment depletes complement within 4 h of the injection, reducing the levels of C3 in the circulation to less than 3 % of the normal range, which persists for at least 48 h post-injection [
33,
34].
RNA isolation and real-time quantitative PCR
Total RNA was extracted from brain tissue using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and was then reversely transcribed using Superscript II (Invitrogen, Carlsbad, CA, USA). Real-time PCR was performed using a SYBR green PCR reagent according to the manufacturer’s instructions (Applied Biosystems, Yerevan, Armenia). The following DNA sequences were used for the primer pairs: P-selectin forward primer, 5'-TCCAGGAAGCTCTGACGTACTTG-3'; P-selectin reverse primer, 5'-GCAGCGTTAGTGAAGACTCCGTAT-3'; E-selectin forward primer, 5‘-TGAACTGAAGGGATCAAGAAGACT-3'; and E-selectin reverse primer, 5'-GCCGAGGGACATCATCACAT-3'; VCAM-1 forward primer, 5‘-TGACAAGTCCCCATCGTTGA-3'; and VCAM-1 reverse primer, 5'-ACCTCGCGACGGCATAATT-3'; intercellular cell adhesion molecule (ICAM-1) forward primer, 5‘-CCTGTTTCCTGCCTCTGAAG-3'; and ICAM-1 reverse primer, 5'-GTCTGCTGAGACCCCTCTTG-3'. Mouse β2-microglobulin (β2-MG) was used as an internal control with the following primer sequences: β2-MG forward primer, 5'-CCTGCAGAGTTAAGCATGACAGT-3'; and β2-MG reverse primer, 5'-TCATGATGCTTGATCACATGTCT-3'. Quantitative PCR was performed with an ABI Prism 7300 spectrofluorometric thermal cycler (Applied Biosystems) using SYBR Green I as a double-stranded DNA-binding dye. The amplification conditions consisted of 95 °C (2 min), followed by 32 cycles of 95 °C (20 s), 57.2 °C (30 s), and 72 °C (30 s). Quantitative PCR assays were conducted in triplicate and were then quantitated using the 2−ΔΔCt method. The data are expressed as n-fold differences relative to the calibrator.
Enzyme-linked immunosorbent assay
The mice were anesthetized after LPS injection and subsequently perfused through the heart with cold PBS to remove blood proteins from the circulation. The brains were rapidly removed and homogenized in 1 ml of sterile PBS, followed by centrifugation at 12,000 rpm for 5 min at 4 °C. The supernatants were assayed for determining TNF-α and IL-1β concentrations using commercial enzyme-linked immunosorbent assay (ELISA) kits (TNF-α, BD, San Diego, CA, USA; IL-1β, eBioscience, San Diego, CA, USA) according to the manufacturers’ instructions. To measure the levels of C3a in brain and plasma of mice, plates were coated with 100 μl of rat anti-mouse capture antibody specifically against C3a (BD Biosciences, San Jose, CA, USA) at 1:250 dilutions in PBS overnight at 4 °C. FUT-175 (BD Biosciences) which was dissolved with 1 ml ddH2O, a synthetic inhibitor for the classical and alternate pathways of complement activation, was added into the EDTA blood and brain samples according to the manufacturer’s instructions. In details, 10 μl of FUT-175 was added into 1 ml of freshly drawn EDTA blood or brain supernatant on ice. After centrifugation, the plasma and brain proteins in these samples were collected for the ELISA assay. Brain, plasma samples, or C3a standard solution were diluted and added to the plates coated with capture antibody and incubated at room temperature for 2 h, and purified mouse C3a protein (BD Bioscience) was set as standard. Brain and plasma samples from C3−/− mice were set as negative control. After washing four times, 100 μl of biotin-conjugated rat anti-mouse C3a (BD Biosciences) at 1:500 dilution in PBS with 10 % FBS was added to each well and incubated at room temperature for 1 h. The wells were washed again and then incubated with a 1:250 dilution of streptavidin/horseradish peroxidase (HRP) (BD Biosciences) for 1 h at room temperature. The wells were washed, and 100 μl of substrate solution (BD Biosciences) was added to each well. The color was developed for 10–20 min with the reaction stopped by the addition of 2N H2SO4. Absorbance was read at 450 nm with correction for absorbance at 550 nm.
Western blotting
The complement factor C3 and albumin were analyzed in the brain homogenates and plasma samples obtained from experimental mice. To obtain the brain homogenates, the mice were anesthetized at 4, 12, and 24 h after i.c.v. LPS injection and subsequently perfused with ice-cold PBS to clear blood-borne proteins. Each brain was removed and homogenized in PBS on ice, followed by centrifugation at 12,000 rpm for 5 min. For the analysis of plasma complement content, blood (100–400 μl) was obtained by intracardiac puncture of anesthetized animals immediately prior to the perfusion. 10 μl of FUT-175 (Futhan) was immediately added into each milliliter of the freshly drawn EDTA blood on ice. After centrifugation at 2500 rpm for 10 min, these plasma samples were boiled in loading buffer for another 10 min. Subsequently, the samples were diluted and separated on a 10 % acrylamide-sodium dodecyl sulfate (SDS) gel, followed by transferring onto membranes and blotting overnight at 4 °C with the following antibodies: anti-C3 (Abcam, Cambridge, USA), anti-albumin (Abcam), anti-E-selectin (Abcam), anti-VCAM-1 (Abcam), anti-ICAM-1 (Abcam), anti-GAPDH (Abcam), anti-NF-kB p65 (Cell Signaling Technology, Beverly, CA, USA), anti-phospho-NF-κB p65 antibody (Cell Signaling Technology), and anti-β-actin (Cell Signaling Technology). The membrane was washed (0.05 % Tween-20 in PBS), incubated with peroxidase-labeled goat anti-rabbit IgG or anti-goat IgG, and washed again. Antibody binding was visualized using enhanced chemiluminescence reagents (PerkinElmer, Waltham, MA, USA). Densitometric images were quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Relative expression levels of proteins were normalized to β-actin.
Isolation and culture of murine cerebral endothelial cells
The mice were sacrificed, and their brains were collected. The cerebral cortices, devoid of cerebella, white matter, and leptomeninges, were minced into small pieces. The pellet was digested in 15 ml of 0.1 % collagenase B (Roche, Indianapolis, IN, USA) supplemented with 30 U/ml of DNase I (Sigma-Aldrich) for 1.5–2 h at 37 °C with occasional agitation. The microvessels were isolated through gradient centrifugation on 15 % dextran (Sigma-Aldrich) and subsequently digested in 0.1 % collagenase/dispase (Roche) supplemented with 20 U/ml of DNase I for 1.5–2 h at 37 °C with occasional agitation. The microvessel pellets were resuspended in medium supplemented with 3 ng/ml of bovine fibroblast growth factor (bFGF, Peprotech, Rocky Hill, NJ, USA), 30 % fetal bovine serum (FBS), 10 U/ml of heparin, 100 U/ml of penicillin, and 100 mg/ml of streptomycin. The microvessel suspension was plated onto 6-well plates coated with rat-tail collagen I (Sigma-Aldrich) and incubated at 37 °C with 5 % CO2. The medium was changed every 2 days. The endothelial cells began migrating from the vessels within 2–3 days and grew to confluence within 7–10 days.
Statistical analysis
Statistical analysis was performed using SPSS software (17.0 for Windows, IBM Inc., Chicago, IL, USA). The values are expressed as the means ± standard errors of the mean (SEM). Differences between the two groups were analyzed using Student’s t test, and P < 0.05 was considered significant.
Discussion
The brain has long been considered to be an immune-privileged organ. However, it is well appreciated that the brain can respond to pathogens and danger signals; in particular, resident glial cells generate inflammatory cytokines and chemokines [
39,
40], which in turn activate endothelial cells to recruit immune cells into the brain [
15]. In CNS inflammation, elevated levels of C3 in CSF in humans and mice with bacterial meningitis have been previously reported [
41,
42]. In addition, several studies have shown critical roles for complement components in brain inflammation and reduced leukocyte recruitment in complement-deficient animals [
43,
44]. However, the detailed mechanism underlying the role of the complement system in immune-cell recruitment into the brain remains unknown. In the present study, we used complement-deficient mice to evaluate the contributions of the complement component C3 in leukocyte recruitment into the brain during inflammation. Our results demonstrated that C3 plays an essential role in cerebral endothelial activation and contributes to neutrophil recruitment into the brain parenchyma.
Complement activation occurs via three different pathways: classical, alternative, and the mannose-binding lectin (MBL) pathways. As the most abundant component of the complement system, C3 is indispensable for all three pathways of complement activation [
45]. The cleavage and activation of C3 initiate the membrane attack pathway, through the formation of the membrane attack complex (MAC), consisting of C5b, C6, C7, C8, and polymeric C9. Neutrophil infiltration into the brain after i.c.v. LPS injection was compromised in C3
−/− mice but not in C6
−/− mice, suggesting that reduced neutrophil recruitment is not dependent on C6 or MAC assembly. The activation of complement system has broad potential biological consequences, because C3a functions as a chemotactic factor for many types of immune cells [
46]. Additionally, we observed significant increased expression of KC(CXCL1) and MIP-2(CXCL2) in the brains of C3
−/− mice (data not shown), and this expression likely provides sufficient chemotactic strength for recruiting neutrophils into the brain parenchyma. Therefore, a lack of chemotactic traction does not explain the inhibition of neutrophil recruitment into the CNS in C3
−/− mice.
Microglial involvement and endothelial activation are two key steps in recruiting neutrophils into the CNS during LPS-induced inflammation. Microglia are the dominant sentinel cells for the detection of bacterial products, and these cells release TNF-α, which activates the endothelium and facilitates leukocyte rolling, adherence, and recruitment into the CNS parenchyma [
47]. In previous studies, we showed that activated glial cells control neutrophil recruitment by secreting TNF-α and CXCL1 [
15,
16]. It has recently been reported that C3 is involved in microglial activation and priming under various CNS inflammatory conditions [
48]. Additionally, complement C1q and C3 are critical for the innate immune response to
Streptococcus pneumoniae in the CNS, and C3
−/− mice were shown to display reduced expression of IL-1β, IL-12 and MIP-1γ in the CNS in a meningitis animal model [
28]. In the present study, only a reduction in the IL-1β level at 24 h was detected in the brain, which is consistent with the findings of previous studies. The levels of KC and MIP-2, essential neutrophil-attracting chemokines, were higher in C3
−/− mice than WT mice at 24 h post-LPS injection (data not shown). In contrast, TNF-α, a key cytokine for the stimulation of endothelial activation and subsequent leukocyte recruitment, did not decrease significantly in C3
−/− animals. Taken together, these results indicate that C3 deficiency did not affect glial cell activation. Upon LPS stimulation, inflammatory cytokines and chemokines were highly produced in these complement-deficient mice, indicating that reduced recruitment might not reflect the compromised glial activation.
The importance of endothelial cell activation in leukocyte recruitment has been well documented [
49,
50]. In a previous study, we reported that a significant increase in TNF-α released from microglia avidly activates the endothelium, causing an increase in adhesion molecule expression and leukocyte recruitment [
15]. Upon LPS injection, the glial cells in C3
−/− mice produced high levels of TNF-α in the brain parenchyma, although significant reduction in leukocyte rolling and adhesion was observed in the brain microvasculature. Accordingly, the expression levels of adhesion molecules (P-selectin, E-selectin, and VCAM-1), which facilitate leukocyte rolling and adherence in brain postcapillary venules, were downregulated in these mice. Clearly, C3 deficiency affected endothelial activation and significantly reduced E-selectin and VCAM-1 expression in C3
−/− animals, even in the presence of high levels of TNF-α, indicating that C3 has synergistic effects with TNF-α on endothelial activation in vivo. C3a stimulates cerebral endothelial activation through its G protein-coupled receptor C3aR. However, TNF-α was able to directly activate endothelial cells to express adhesion molecules in the absence of C3. The mechanism for this discrepancy between the in vivo and in vitro observations remains to be elucidated.
The liver is a primary source of complement components [
51], most of which remain within the circulation. Additionally, it has been reported that the local synthesis of complement components in resident cells in the CNS plays an essential role during CNS inflammation [
52,
53]. The complement components detected in the CNS are primarily derived from two sources: (1) complement components are locally synthesized from different types of neural glial cells in the brain [
54,
55]; and (2) the BBB is not impermeable, and complement components can bypass the barriers and penetrate into the brain parenchyma within 12 h after BBB integrity has been compromised. C3 was only detected in the CNS 12 h after LPS injection. Thus, considering that endothelial cell activation and leukocyte rolling and adhesion were activated at 4 h post-injection, when the C3 level in the brain homogenate was nearly undetectable, C3 from the brain parenchyma was unlikely to facilitate endothelial activation and leukocyte-endothelial interactions. We propose that complement components from the circulation may play a major role in endothelial activation for the following two reasons. First, endothelial activation was dependent on the expression of adhesion molecules on the CNS endothelium. Notably, most of the adhesion molecules were expressed on the luminal side of the CNS endothelium. Thus, it would be easier for C3 in the circulation, rather than factors secreted from the CNS, to mediate the expression of adhesion molecules on the luminal side of the brain endothelium. Second, C3 levels in the circulation were much higher than those in the brain, even after a 100-fold dilution of the plasma samples. Additionally, CVF depletes the complements in the circulation via C3 activation and cleavage. This could potentially cause a massive increase in C3a levels. However, the increase of C3a might not last for a long period and might be depleted from circulation in a very short time. We have already measured levels of C3a at 4 h (the time point for endothelial activation) and 8 h after CVF injection and detected minimum amount of C3a in the circulation. Further, CVF treatment significantly reduced brain endothelial activation. Taken together, these results indicate that it is the C3 in the circulation that facilitates brain endothelial activation.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
FJW and DYS performed the intravital microscopy and histology experiments. DXD and FJW performed the quantitative PCR and Western blotting experiments. XXZ conducted the cell cultivation experiments. HZ and QZ conceived the study. HZ wrote the manuscript, and LL and WGH revised the manuscript for important intellectual content. All authors read and approved the final manuscript.