Institut Fédératif de Recherches Multidisciplinaires sur les Peptides no. 23, INSERM U78, Faculté de Médecine et Pharmacie, 22 Boulevard Gambetta, 76183 Rouen Cedex, France
1 INSERM U413, Université de Rouen, 76821 Mont-Saint-Aignan Cedex, France
Correspondence to: M. Fontaine
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Abstract |
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Keywords: brain, complement, differentiation, expression, inflammation, neuroimmunology, neuron, regulation
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Introduction |
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Although the liver is the primary site of synthesis of complement components, numerous extra-hepatic sources of complement have been shown. These includes monocytes/macrophages (3,4), fibroblasts (5,6), epithelial cells (79), keratinocytes (10,11), endothelial cells (1215), adipocytes (16,17), osteoblastic cells (18,19) and skeletal myoblasts (2022). This extra-hepatic synthesis of complement plays an important role in local inflammatory processes but complement presents new functions which are not connected with immunity and which are specific of the tissue where complement is expressed (for review, see 23).
Evidence implicating complement in demyelination, neurodegeneration and other central nervous system (CNS) pathologies has accumulated during the last few years (for review, see 24). Cells in the brain are normally protected from serum complement by the bloodbrain barrier, but complement may be synthesized intrathecally, raising the possibility that endogenous complement might damage surrounding neurons and oligodendrocytes, and contribute to pathology in inflammatory CNS diseases. Complement biosynthesis in the CNS is now well established. Microglia (2527), astrocytes (2833) and oligodendrocytes (34) are all sources of complement in the brain. Moreover, neurons have been suggested to be a potential source of complement. Complement C4 mRNA was detected by in situ hybridization in neurons in a model of experimental rat brain lesioning (25,26). Walker and McGeer detected C4 and C3 mRNA in neuroblastoma cell lines by RT-PCR (35), and expression of the classical complement pathway was recently observed by in situ hybridization and immunohistochemistry in post-mortem Alzheimer's disease brain tissue (36,37).
It is quite difficult to obtain and maintain pure cultures of neurons from human brain. To get round this problem, well-characterized human neuroblastoma cell lines can be used as a model for human neurons. Using this model, we have previously shown that human neuroblastoma cells have the capacity to spontaneously activate the classical pathway of complement in serum, but with moderate cell killing, despite the deposition of activation complement fragments on these cells (38). Neuroblastoma abundantly expressed complement inhibitors membrane cofactor protein (MCP) and CD59, but not decay accelerating factor (DAF) and complement receptor 1 (CR1) (38).
To enlarge the relationship between neurons and the complement system, we wanted to observe if neurons had the capacity to express a complete complement system. In this paper, we demonstrate the expression of components of the alternative, classical and terminal pathways of the complement system, i.e. C3, factor H (FH), factor B (FB), C4, C1-inhibitor (C1-inh), C1q, C5, C6, C7 and C9, by neuronal cells and their regulation by different factors.
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Methods |
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Cell cultures
Three human neuroblastoma cell lines (IMR32, SKNSH and KELLY) were obtained from ATCC (Rockville, MD). Human neuroblastoma cell line SH-SY5Y was generously provided by Dr A. Delacourte (INSERM U422, Lille, France). Cells were cultivated in DMEM medium (Sigma) containing 10% heat-inactivated FCS (Sigma), 2 mM L-glutamine (Biowhittaker, Fontenay-sous-Bois, France), 100 IU/ml penicillin (Biowhittaker) and 100 µg/ml streptomycin (Biowhittaker) in 75 cm2 flasks (Falcon, Poly-Labo, Strasbourg, France), and grown at 37°C in a humidified incubator containing 5% CO2/95% air. The culture medium was changed every 48 h. Cells were dispersed for culture passages with 0.05% trypsin/0.02% EDTA in PBS without Ca2+/Mg2+ (Sigma). Cells were then centrifuged at 200 g and the pellets were dispersed in the culture medium. The cell lines were routinely screened by the Hoechst 33258 DNA staining method to ensure that they were mycoplasma-free.
Cells were stimulated with different concentrations of cytokines (201000 IU/ml), DXM (107105 M) and LPS (0.55.0 µg/ml) for various periods (672 h). For quantification in all experiments, cells were removed by gentle scraping or by brief trypsinization and counted.
Immature granule cells were obtained from cerebelli of 8-day-old Wistar rats and cultured in a chemically defined medium as previously described (39). Differentiated granule cells were cultured in a DMEM medium supplemented with 25% Ham's F12 medium, 10% FCS, 25 mM KCl and 1% of an antibioticantimicotic solution (10000 U/ml penicillin, 10 mg/ml streptomycin and 25 µg/ml fungizone). Then 10 µM cytosine-arabinofuranoside (Sigma) was added in the culture medium 24 h after plating, in order to eliminate non-neuronal cells. Differentiated granule cells were used after 10 days in vitro.
Western blotting experiments
Confluent cells were intensively washed with apyrogen 0.9% NaCl and cultivated in DMEM medium without FCS for 48 h. Supernatants were collected, centrifuged at 400 g for 10 min to remove cellular debris, mixed with a cocktail of proteinase inhibitors (1 mM EDTA, 1 mM EGTA, 5 mM benzamidine, 10 µg/ml leupeptin and 5 µg/ml pepstatin A), dialysed overnight against buffer (50 mM Tris/10 mM EDTA) and concentrated 20 times by lyophilization. The powder was dissolved with 1 volume of distilled water and 1 volume of Laemmli buffer (2% SDS, 10% glycerol and 250 mM TrisHCl, pH 6.8). This concentrate was analyzed by SDSPAGE according to the method of Laemmli (40) in non-reducing conditions. Then, the proteins were electrotransferred onto a Biotrace nitrocellulose sheet (Gelman, Ann Arbor, MI) at 6 V/cm for 2h, as described by Towbin et al. (41). Prestained protein markers from Sigma were used as mol. wt standards.
Blots were saturated in 0.2% BSA/0.05% gelatin in PBS and then incubated with a specific polyclonal biotinylated antibody diluted in saturation buffer. After extensive washings, the peroxidase-conjugated avidin diluted in saturation buffer was added to the nitrocellulose sheet. Finally, the blots were developed with the enhanced chemiluminescence (ECL) Western blotting detection system (Amersham, Les Ulis, France).
ELISA
Cells were cultivated on 24-well plates (Falcon), and, after reaching confluence, washed with 0.9% NaCl and 500 µl of DMEM medium with 10% FCS, with or without stimulating factors, added per well. The whole supernatant from unstimulated or stimulated-cells were collected at 6, 12, 24, 30, 48 and 72 h, treated with a mixture of proteinase inhibitors, and frozen at 70°C until used.
Complement proteins were quantified in cell culture supernatants by ELISA in 96-well microtiter plates (Virion, Roche Products, Rosny-sous-Bois, France), as previously described (20). For each assay, a standard curve was established with known amounts of a calibrated pool of normal human sera.
Statistical analysis
The significance of difference between values was estimated by Student's t-test. P < 0.05 was considered to show statistically significant differences.
RNA preparation and RT-PCR
Total RNA from culture of neuroblastoma cell lines was obtained by the guanidium isothiocyanate procedure (42) and ultracentrifugation on cesium chloride gradients. Total RNA from rat cerebellum granular primary cell culture was prepared using the RNeasy extraction kit according to the manufacturer's instructions (Qiagen, Courtaboeuf, France). The purity and yield of total RNA were determined spectrophotometrically by measuring the absorbance of an aliquot at 260 and 280 nm. RNA preparation was checked by agarose gel electrophoresis.
RT-PCR was performed according to the protocol of Kawasaki (43), using 2 µg of total RNA, primer pd(N)6 (Pharmacia, St Quentin-en-Yvelines, France), and MMLV reverse transcriptase (Gibco/BRL, Life Technologies, Cergy-Pontoise, France) for the reverse transcription reaction and specific 20mer oligonucleotide primers (Genosys, Cambridge, UK) for PCR in a Hybaid Omnigene (Céra-Labo, Aubervilliers, France) DNA thermal cycler (30 cycles) (detailed protocol on request). Amplification of specific cDNA was confirmed on agarose gels staining with ethidium bromide for UV detection.
Different controls were performed (no RNA, no MMLV reverse transcriptase or no reverse transcriptase product) to ensure the validity of the reaction product. Primer positions (upstream and downstream) are given according to their position (nucleotide number in the cDNA sequence from 5' to 3'). The EMBL accession numbers, amplified sequences and expected product sizes were as follows: C3: K02765, 24562690, 234 bp; FH: X07523, 334826, 492 bp; FB: K01566, 10321590, 558 bp; C4: K02403, 515772, 257 bp; C1-inh: M13203, 51401, 350 bp; C1q: X03084, 94531, 437 bp; C5: M57729, 39014300, 399 bp; C6: J05024, 20412437, 396 bp; C7: J03507, 24012790, 389 bp; C9: X02176, 13611771, 410 bp; rat C3: M29866, 172609, 437 bp; rat C1q: X71127, 333792, 459 bp.
Functional tests
Functional activities of C3 and FH were determined as described (31). Briefly, C3 activity was estimated by hemolytic C3 assay. Activities of FH were investigated by their capacity to cleave the chain of C3b (cofactor assay). The cleavage of 125I-labeled C3b by FH was analyzed by electrophoresis and autoradiography. The percent of residual C3b
chain was estimated by scanning autoradiograms using a Lecphor camera densitometer coupled to a computer (Biocom, Les Ulis, France).
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Results |
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The protocol used for concentration of supernatants precluded an accurate quantitative analysis of these data. Quantitative effects of cytokines on secretion were studied in detail by ELISA (see below).
Functional activity of C3 and FH produced by SKNSH cells
The functional activity of the newly secreted C3 was investigated by hemolytic assay. The sp. act. of C3 produced by SKNSH cells was estimated to be 1.3 ± 0.5x105 effective molecules/ng of C3. The specific activity of fresh normal serum was determined in parallel and is similar to that of C3 from SKNSH cells (not shown).
FH purified from supernatants of SKNSH cells was functionally active as demonstrated by its capacity to cleave C3b. Anti-FH antibody completely blocked this cleavage (not shown).
Effects of cytokines on regulation of C3, FH, FB, C4 and C1-inh secretion by SKNSH cells
SKNSH cells were cultivated in medium containing different amounts of stimulating factors: IFN-, IL-1ß, TNF-
, IL-6, LPS and DXM. We determined the kinetics of C3, FH, FB, C4 and C1-inh secretion by ELISA.
Two cytokines appeared to regulate significantly the secretion of FH and C1-inh by SKNSH cells: (i) IFN- which increased FH (8-fold; P < 0.0001) and C1-inh (4-fold; P < 0.0001) secretion, and (ii) TNF-
which increased FH (2.5-fold; P < 0.0001) and C1-inh (4-fold; P < 0.0001) secretion (Fig. 3
). These effects were time dependent with a maximum effect at 48 h and dose dependent (not shown).
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To observe the conjugated effects of cytokines on FH and C1-inh secretion by SKNSH cells, different combinations of cytokines were made (Fig. 3).
Combinations of these different stimulating factors had no significant effects on complement proteins secretion compared to the effects of the cytokine alone. In a general manner, IFN- and TNF-
were very slightly enhanced by IL-1ß. The combination of TNF-
and IFN-
gave the higher rate of secretion.
Qualitative analysis of mRNA expression of complement components by SKNSH cells and other neuroblastoma cell lines by RT-PCR
Total RNA were purified from SKNSH cell culture. After reverse transcription, PCR was performed with specific primers to analyze the expression of C3, FH, FB, C4 and C1-inh mRNA. PCR products were electrophoresed on 1% agarose gels in parallel with positive and negative controls of the RT-PCR reactions.
SKNSH cell line expressed mRNA for C3, FH, FB, C4 and C1-inh since amplification products were identified at the correct size by comparison with size markers. Expression of C3, FH, FB, C4 and C1-inh mRNA was detected under basal conditions and after stimulation with 200 IU/ml IFN- (Table 1
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Expression of these complement components mRNA was studied from the others neuroblastoma cell lines IMR32, KELLY and SH-SY5Y under the same conditions (Table 1). SH-SY5Y cells expressed constitutively and under stimulation C3, FH, FB, C4, C1-inh, C1q, C5, C6, C7 and C9 mRNA. In IMR32 cell culture, the expression of FH, C4, C1-inh, C1q, C6 and C9 mRNA was constitutive and easily detectable, whereas that of C3 mRNA was very low, C5 mRNA was induced by IFN-
, and FB and C7 mRNA were never detected. In KELLY cell line culture, the expression of FH, C4, C1-inh, C1q and C9 mRNA was detected constitutively and under stimulations, C6 mRNA was induced by IFN-
stimulation, and C3, FB, C5 and C7 mRNA were not detected.
These results of mRNA expression observed by RT-PCR have been confirmed by studies on effects of cytokines on complement components secretion by the four cell lines, measured by ELISA (not shown). Moreover, it appears from this last study that each cell line synthesized each protein at its own rate, IMR32 cells being the lowest potent complement producer, and that the four cell lines exhibited the same response to cytokines.
Analysis of complement components mRNA expression by rat cerebellum granular primary cell culture by RT-PCR
To investigate complement expression by normal neurons in vitro, total RNA was purified from undifferentiated or differentiated rat cerebellum granular primary cell culture. After reverse transcription, PCR was performed with specific primers to analyze the expression of C3, C4 and C1q mRNA. PCR products were electrophoresed on 1% agarose gels in parallel with positive and negative controls of the RT-PCR reactions (Fig. 4).
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Discussion |
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Previous observations (25,26,3537) mainly related to the expression of mRNA of C4 and C3 or of the classical pathway during Alzheimer's disease have suggested that cells of neuronal origin have the capacity to express complement components. Our previous works have shown that human neuroblastoma cells have the capacity to spontaneously activate the classical pathway of complement in serum and abundantly expressed complement inhibitors MCP and CD59, but not DAF and CR1 (38). This study described for the first time the expression and regulation of complement proteins by neuronal cells. Using cultured human neuroblastoma cell lines, we clearly demonstrate the expression of three components of the alternative pathway and three of the classical pathway of the complement system: C3, FH, FB, C4, C1q and C1-inh. These syntheses were detected by Western blotting and quantified by ELISA, performed on cell supernatants. Specific mRNA for C3, C4, C1q, FB, FH and C1-inh were demonstrated by RT-PCR, and proved the neuronal origin of these syntheses in vitro. Moreover, specific mRNA for C5, C6, C7 and C9 was also demonstrated by RT-PCR. These results showed that neuronal cells can produce a complete complement system.
The newly synthesized proteins were similar in molecular size and activity to their serum counterparts. For FH, an additional band of 43 kDa was observed in addition to the well-characterized 155 kDa species when analyzed by SDSPAGE under non-reduced conditions. This 43 kDa species most probably corresponds to the N-terminal truncated form found in plasma (44,45).
Thus, these cells represent interesting models easily available to analyze complement expression by neurons, because they produce two functional activation pathways and, with the synthesis of terminal components, probably lead to the formation of the cytolytic membrane attack complex.
To investigate complement expression by normal neurons in vitro, we analyzed the expression of C3, C4 and C1q mRNA. Undifferentiated rat cerebellum granular primary cells expressed constitutively mRNA for C4 and C1q but no C3 mRNA.
Synthesis of complement components by neuronal cells had a specific feature compared to other cell types producing complement (i.e. macrophages, endothelial cells or glial cells). The most prominent characteristic was the inefficacy of IFN- or other cytokines to stimulate the synthesis of C3, C4 and FB. Only synthesis of regulatory proteins FH and C1-inh was highly up-regulated by IFN-
and TNF-
. This would indicate that neurons respond to inflammation by increasing the secretion of complement regulators to inhibit complement activation.
Another feature but shared by neurons and by monocytic cells was a correlation between the basal rate of complement biosynthesis and the degree of differentiation of neurons. There were significant variations between the basal rate of complement synthesis by the four cell lines studied. These differences may be attributed to the degree of differentiation of the cells because the IMR32 cell line, which was the less differentiated cell line, had the lowest rate of complement components synthesis. However, the four cell lines responded identically to cytokine stimuli. This effect of differentiation was also observed on normal rat neurons. Rat cerebellum granular cells expressed constitutively mRNA for C4 and C1q, but not C3 mRNA. Differentiation of these cells in mature neurons, not only enhanced C4 and C1q mRNA expression, but induced mRNA expression of C3.
The role of complement expression by neurons remains unclear. Complement has also been implicated in several brain disorders, notably demyelination and neurodegeneration. During Alzheimer's disease, an increase of complement components mRNA was observed and recently was attributed to neurons by in situ hybridization and immunohistochemistry (36,37). Our in vitro data showed that cytokines were not involved in this phenomenon since C3 and C4 synthesis were not up-regulated by inflammatory cytokines (TNF-, IL-1ß, IL-6 and IFN-
), suggesting that other factors were responsible for complement overexpression during the disease, possibly ß-amyloid peptide that was previously shown to enhance C3 expression by microglia (27). During Alzheimer's disease, complement could participate to neuron loss by different pathways. We previously showed that complement could kill neurons by direct lysis (38). Recently, Farkas et al. (46) induced a cell death program in neurons by a C5a-derived peptide, suggesting that anaphylatoxins may trigger neuron death. The recent observations of anaphylatoxin receptor expression by neurons (47) highlight the biological signification of complement in the brain.
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Acknowledgments |
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Abbreviations |
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C1-inh C1-inhibitor |
CNS central nervous system |
CR1 complement receptor 1 |
DAF decay accelerating factor |
DXM dexamethasone |
FB factor B |
FH factor H |
LPS lipopolysaccharide |
MCP membrane cofactor protein |
TNF tumor necrosis factor |
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Notes |
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Transmitting editor: A. Fischer
Received 10 May 1999, accepted 15 March 2000.
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References |
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