Human oligodendroglial cells express low levels of C1 inhibitor and membrane cofactor protein mRNAs
© Hosokawa et al; licensee BioMed Central Ltd. 2004
Received: 20 May 2004
Accepted: 24 August 2004
Published: 24 August 2004
Oligodendrocytes, neurons, astrocytes, microglia, and endothelial cells are capable of synthesizing complement inhibitor proteins. Oligodendrocytes are vulnerable to complement attack, which is particularly observed in multiple sclerosis. This vulnerability may be related to a deficiency in their ability to express complement regulatory proteins.
This study compared the expression level of complement inhibitor mRNAs by human oligodendrocytes, astrocytes and microglia using semi-quantitative RT-PCR.
Semi-quantitative RT-PCR analysis showed that C1 inhibitor (C1-inh) mRNA expression was dramatically lower in oligodendroglial cells compared with astrocytes and microglia. The mRNA expression level of membrane cofactor protein (MCP) by oligodendrocytes was also significantly lower than for other cell types.
The lower mRNA expression of C1-inh and MCP by oligodendrocytes could contribute to their vulnerability in several neurodegenerative and inflammatory diseases of the central nervous system.
Resident brain cells including oligodendrocytes [1, 2], astrocytes, astrocytomas, microglia, glioblastomas [3–14], neurons [15, 16], neuroblastomas [17, 18] and endothelial cells [19, 20] express mRNAs for complement proteins. Although the role of complement expression by these cells remains unclear, local complement activation in the central nervous system (CNS) might damage these cells and contribute to the pathology in several inflammatory and neurodegenerative diseases including multiple sclerosis, Alzheimer's disease and progressive supranuclear palsy.
For self-protection, resident brain cells also express complement inhibitors, such as membrane cofactor protein (MCP), decay-accelerating factor (DAF), CD59, and C1-esterase inhibitor (C1-inh). The human HOG oligodendroglial cell line produces MCP, DAF, CD59, C1-inh and S-protein, but not complement receptor 1 (CR1) . Human oligodendrocytes have been reported to express CD59  and DAF, but not MCP, CR1, homologous restriction factor (HRF: C8 bp) or clusterin . Astrocytes , neurons and Schwann cells have been reported to express CD59  and neuroblastoma cell lines C1-inh . Astrocytoma cell lines have been reported to express MCP, DAF, and CD59 [25, 26].
In this study, the expression level of mRNAs for various complement inhibitors by human oligodendrocytes, astrocytes and microglia were compared by semi-quantitative PCR. We show that oligodendrocytes express extremely low levels of mRNA for C1-inh and significantly lower levels of mRNA for MCP compared with astrocytes and microglia. The expression level of mRNAs for CD59 and DAF showed no significant differences between the three cell types.
Cell culture: microglial- and astrocyte-enriched cultures
Human microglial and astrocytic cells were isolated from surgically resected temporal lobe tissues. We thank Dr. J. Maguire, Department of Pathology and Laboratory Medicine, Vancouver General Hospital for providing the surgical specimens. Isolation protocols described by De Groot et al. [27, 28] were used with minor modifications. Tissues were placed in a sterile Petri dish, rinsed with Hank's balanced salt solution, and visible blood vessels were removed. After washing tissues two more times with Hank's balanced salt solution, tissues were chopped into small (<2 mm3) pieces with a sterile scalpel. The fragments were transferred into a 50 ml centrifuge tube containing 10 ml of 0.25% trypsin solution (Gibco-BRL, Life Technologies, Burlington, ON, Canada), and incubated at 37°C for 20 min. Subsequently DNase I (from bovine pancreas, Pharmacia Biotech, Baie d'Urfé, PQ, Canada) was added to reach a final concentration of 50 μg/ml. Tissues were incubated for an additional 10 min at 37°C. The cell suspension was diluted with 10 ml of Dulbecco's modified Eagle's medium (DMEM) and nutrient mixture F12 ham (DMEM-F12; Sigma-Aldrich, Oakville, ON, Canada) with 10% fetal bovine serum (FBS; Gibco-BRL, Life Technologies), and gently triturated by using a 10 ml pipette with a wide mouth. After centrifugation at 275 × g for 10 min, the cell pellet was resuspended in serum containing medium, triturated several times, and passed through a 100 μm nylon cell strainer (Becton Dickinson, Franklin Lakes, NJ). The cell suspension was then centrifuged once more (275 × g for 10 min), resuspended into 10 ml of DMEM-F12 with 10% FBS containing gentamicin (50 μg/ml, from Sigma), and plated onto uncoated 10 cm tissue culture plates (Becton Dickinson). Plates were placed in a humidified 5% CO2, 95% air atmosphere at 37°C for 2 hr in order to achieve adherence of microglial cells. Non-adherent cells with myelin debris were removed from these microglia-enriched cultures and transferred into poly-L-lysine coated 10 cm tissue culture plates in order to achieve adherence of astrocytes. Plates were incubated for 48 hr, after which the culture medium containing myelin debris and non-adherent cells was removed and used to prepare oligodendroglial cell cultures as described below. Both microglial- and astrocyte-enriched cultures were grown for 6 to 7 days before their mRNAs were extracted. Immunostaining with antibodies against CD68 (Dako, Mississauga, ON, Canada) which stains microglia as well as macrophages, and glial fibrillary acidic protein (GFAP, Dako), which is a marker of astrocytes, showed that the microglia-enriched cultures contained 93.5 ± 3.6 % (N = 4) microglial cells, while astrocyte-enriched cultures contained 85.7 ± 3.4 % (N = 4) astrocytes.
Cell culture: oligodendroglial cells
These were prepared as described before . Briefly, cell culture media containing myelin debris and non-adherent cells that were removed from astrocyte-enriched cultures were used to extract oligodendroglial cells. The non-adherent cells were collected by centrifugation at 275 × g for 10 min and replated onto uncoated 10 cm tissue culture plates for another 24 hr. Subsequently, the cell culture medium containing floating cells was transferred to 50 ml tubes and Lymphoprep solution (Axis-Shield, Oslo, Norway) used to reduce the amount of contaminating myelin debris. For this purpose, 10 ml of Lymphoprep solution was carefully placed under the oligodendrocyte cell suspension and the density gradient was centrifuged at 325 × g for 10 min. The interphase was collected and transferred to a 50 ml centrifuge tube. Fresh culture medium was added and the suspension was centrifuged at 275 × g for 7 min. The cell pellet was resuspended and the oligodendrocyte cultures seeded onto 60 mm plastic culture dishes. Immunostaining with anti-O4 antibody (Chemicon International, Temecula, CA), which is a marker of oligodendrocytes, showed that the oligodendrocytes-enriched cultures contained 95.3 ± 4.4 % (N = 4) oligodendrocytes.
RNA isolation and cDNA synthesis by reverse transcription
Total RNA from oligodendroglial cells, microglia, and astrocytes were isolated by the acid guanidium thiocyanate-phenol-chloroform method. Two μg of the RNA was then used to prepare cDNA. RNA was treated with 10 U of DNase I (Gibco BRL, Life Technologies) for 60 min at 37°C in 25 μl of 1 × reverse transcriptase buffer (50 mM Tris-HCl, 75 mM KCl, 3 mM MgCl2) containing 40 U of RNase inhibitor (Pharmacia Biotech) and 1 mM dithiothreitol (DTT), following by incubation at 85°C for 5 min to inactivate the enzyme. Reverse transcription was performed at 42°C for 90 min in 50 μl of the following mixture: 1 × reverse transcriptase buffer containing 2 μg of RNA, 5 mM DTT, 0.2 μg random hexamer primers (Pharmacia Biotech), 1 mM deoxynucleotides (Gibco BRL, Life Technologies), 40 units of RNase inhibitor, and 400 units of SuperScript II reverse transcriptase (Invitrogen Life Technologies, Burlington, ON, Canada). At the end of the incubation period, the enzyme was inactivated by heating at 65°C for 10 min .
Polymerase chain reaction
Oligonucleotide primers used for PCR, and the corresponding restriction endonucleases used for product confirmation.
Sequence (5' → 3')
Fragment size (introns)
Genbank accession No
Restriction enzymes used and the expected sizes of digestion products (bp)
GTT GGG GGA TGC TTT GGT AGA TTT C
Sau 3AI (246, 86)
TTA GGA CTC TGG GGC TGC TGC TGT A
CTG CTG CTC GTC CTG GCT GTC TTC T
Pst I (233, 47)
TCC CAC CAT TTT CAA GCT GTT CGT T
CAA TTC AGT GTG GAG TCG TGC TGC
Sau 3AI (193, 72)
TGA GGC ACT GGA CGC TGG AGA T
GTA CTG TGA ATA ATG ATG AAG GAG
Hae III (330, 34)
TCT TAA CTC TTC TTT GGC TAA GTC
CCA TGT TCG TCA TGG GTG TGA ACC A
Dde I (168, 83)
GCC AGT AGA GGC AGG GAT GAT GTT C
PCR primer design and restriction analyses
Primers were designed to span introns so that cDNA-derived PCR products would be of different sizes to those produced if genomic DNA was amplified (see Table 1). DAF and MCP were exceptions, since only cDNA sequences were available. Primers were synthesized either by Sigma-Aldrich or ID Labs (London, ON, Canada). The primer sequences and predicted PCR fragment sizes are listed in Table 1, along with the names of the enzymes used for restriction digest analysis of each PCR fragment. The restriction digestion reactions were carried out at 37°C for 2 hr in the presence of 1 × the appropriate buffer provided by the suppliers (Invitrogen, Life Technologies and New England Biolabs, Mississauga, ON, Canada). The digested PCR products were analyzed on a 6% polyacrylamide gel (data not shown). In all cases the restriction fragments observed were of the predicted size (see Table 1).
The data are presented as means ± s.e.m. The significance of difference between values was estimated by means of one-way analysis of variance (ANOVA) with Fisher's LSD post-hoc test. P < 0.05 was considered to show statistically significant differences.
Double fluorescence immunocytochemical analysis
Oligodendrocytes, astrocytes, and microglia were harvested and air-dried on glass slides. Cells were then fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 5 min. For inactivation of endogenous peroxidase, cells were incubated with 0.3% H2O2 for 30 min. Blocking was performed for 1 hr at room temperature in 5% skim milk.
For double fluorescence immunostaining, cells were incubated at room temperature overnight with a primary antibody in 1% normal serum. The primary antibody and the dilution used in the first cycle were as follows: O4 (Chemicon International, 1: 100) for oligodendrocytes, GFAP (Dako, 1: 10,000) for astrocytes, CD68 (DAKO, 1: 50) for microglia. Cells were then treated for 2 hr at room temperature with a biotin conjugated anti-mouse IgM (Vector Laboratories, Burlingame, CA, 1: 200) secondary antibody for O4, a biotin conjugated anti-rabbit IgG (Vector Laboratories, 1: 200) secondary antibody for GFAP and a biotin conjugated anti-mouse IgG (Vector Laboratories, 1: 200) secondary antibody for CD68. Then cells were incubated with Texas Red Avidin DCS (Vector Laboratories) for 1 hr. The primary antibody and the dilution used in the second cycle were as follows: for C1-inh, goat anti-C1-inhbitor (Quidel, San Diego, CA, 1: 50); for CD59, mouse anti-CD59 (Serotec Ltd, Oxford, UK, 1: 10) or rat anti-CD59 (Serotec, 1: 25). Cells were incubated at 4°C for 3 days with a primary antibody in 1% serum corresponding to the secondary antibody type. Cells were then treated for 2 hr at room temperature with FITC-conjugated anti-mouse IgG (Vector Laboratories, 1: 200), anti-goat IgG (Santa Cruz Biotechnology, Santa Cruz, CA, 1: 200), or anti-rat IgG (Cappel, Durham, NC, 1: 200). The glass slides were then rinsed with distilled water, and a drop of Vectashield mounting medium (Vector Laboratories) placed on the slide.
Semi-quantitative RT-PCR analysis
Double fluorescence immunohistochemistry
This report shows that human oligodendrocytes express a much lower level of mRNA for C1-inh than astrocytes and microglia, and a significantly lower level of mRNA for MCP. The mRNA levels of CD59 and DAF were comparable in all the three cell types. Overall our data suggest that oligodendroglial cells, in common with other cell types, can produce complement inhibitors, but at a significantly lower level for C1-inh and MCP.
It has already been reported that human neurons and Schwann cells , neuroblastoma cell lines , astrocytes , astrocytoma cell lines [25, 26], the HOG human oligodendroglial cell line  and oligodendrocytes [21, 22] produce some or all of the complement inhibitor proteins and their mRNAs.
Activation of the complement cascade and deposition of activated complement fragments occur in non-infectious diseases such as multiple sclerosis, Pick's disease, Alzheimer's disease and other neurodegenerative conditions [15, 16, 30–34]. Complement inhibitors may play an important role in preventing such pathology.
Full activation of the complement cascade requires overcoming a series of endogenous inhibitory factors. Oligodendrocytes are vulnerable to complement attack, which is particularly observed in multiple sclerosis [35–37] and this vulnerability may be related to a deficiency of their ability to express complement regulatory proteins , particularly C1-inh.
Sporadic complement attack, in the form of complement activated oligodendroglia (CAO) is also observed in a number of neurodegenerative conditions [38, 39], including Alzheimer's, Pick's, Huntington's and Parkinson's diseases, amyotrophic lateral sclerosis, progressive supranuclear palsy, Shy-Drager syndrome, argyrophilic grain dementia and pallido-nigral luysial atrophy [38, 39]. The source of the complement proteins that become activated is unknown, but the data presented here suggest that oligodendrocytes are vulnerable to complement attack because of a low expression of C1-inh and MCP.
These results suggest that the lower expression of C1-inh and MCP by oligodendrocytes could contribute to their vulnerability in several neurodegenerative and inflammatory diseases of the central nervous system, particularly multiple sclerosis.
List of abbreviations
analysis of variance
central nervous system
complement activated oligodendroglia
complement receptor 1
fluorescein isothiocyanate isomer
glial fibrillary acidic protein
homologous restriction factor
membrane cofactor protein
This work was supported by a grant from the Jack Brown and Family Alzheimer's Disease Research Fund, and the Pacific Parkinson's Research Institute.
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