Human Molecular Genetics, 2004, Vol. 13, No. 3
DOI: 10.1093/hmg/ddh029
Advance Access published on December 8, 2003
295–302
Expression of complement components in
the peripheral nervous system
Rosalein R. de Jonge1, Ivo N. van Schaik2, Jeroen P. Vreijling1, Dirk Troost3
and Frank Baas1,2,*
1
Neurogenetics Laboratory, 2Department of Neurology and 3Department of Pathology, Academic Medical Center,
Amsterdam, The Netherlands
Received August 28, 2003; Revised November 15, 2003; Accepted November 27, 2003
We have generated a SAGE (serial analysis of gene expression) library of normal sciatic nerve and found tags
encoding for mRNAs of the complement system highly represented. RNA (RT–PCR and northern blot
hybridization) and protein (western blot analysis and immunohistochemistry) studies confirmed these
findings. High expression of classical pathway components, alternative pathway components and inhibitory
components was observed in specific regions of the sciatic nerve. The first components of complement were
found in axons, whereas the inhibitory components were detected in the perineurium, thereby protecting the
nerve from a complement attack. Immunoreactivity towards activated complement factors was noted in post
traumatic neuromas and after acute crush injury, which exemplify nerve regeneration and degeneration. We
propose that local production of complement in the peripheral nervous system participates in the protection
of healthy nerve and is needed for efficient clearance of myelin after injury: a prerequisite for normal
regeneration and remyelination of the peripheral nerve.
INTRODUCTION
The complement system plays a major role in host defence
against microorganisms and in the processing and elimination
of immune complexes. The complement system consists of
some 30 proteins, which include soluble as well as membraneembedded complement proteins (1). Three distinct routes, the
classical, the alternative and the lectin pathway can activate
complement and lead to the formation of the C5b–C9 cytolytic
membrane attack complex (MAC) (2,3). The primary site of
synthesis of the majority of the plasma complement proteins is
liver. Extra-hepatic complement biosynthesis, known to occur
in several tissues, may be an important factor in triggering and
perpetuating local inflammatory reactions, especially in tissues
that are shielded from plasma components by a blood–tissue
barrier (4,5).
The human brain, which is protected by the blood–brain
barrier, is an example of an organ with its own local
biosynthesis of the complement system (3,6). Complement
has been implicated in several neurodegenerative diseases of
the brain, like Alzheimer’s disease, Huntington’s disease and
Pick’s disease (6). Complement activation is also seen in
immune-mediated neurological disorders such as multiple
sclerosis (7). In the peripheral nervous system (PNS), several
types of neuropathy are suspected to be autoimmune in origin
and circulating autoantibodies to myelin and Schwann cell
antigens have been detected (8–16). Complement is implicated
as an effector in inflammatory demyelination observed in
experimental allergic neuritis (EAN), a model for Guillain–
Barré syndrome, an immune-mediated acquired human demyelinating neuropathy (17). In patients with polyneuropathy and
IgM monoclonal gammopathy, deposition of several complement components and of the MAC on the myelin sheaths of
peripheral nerves has been reported (18).
Complement proteins have also been implicated in Wallerian
degeneration (19). Transection of axons in the PNS leads to a
pattern of distal axonal degeneration, followed by myelin
degradation and Schwann cell and fibroblast proliferation.
Macrophages participate in the cellular responses during
Wallerian degeneration, and although the exact mechanism
for their recruitment is not completely understood, complement
is believed to play a role. Serum C3-depleted rats show a
reduced macrophage infiltration and a reduced capacity to clear
myelin (20), whereas C5 deficient mice show a delay in
macrophage recruitment as well as axonal breakdown and
myelin sheath elimination after sciatic nerve crush (21).
Local synthesis of complement components in human
peripheral nerve has thus far not been shown. In this study,
*To whom correspondence should be addressed at: Neurogenetics Laboratory, Academic Medical Center, K2-213, PO Box 22660, 1100 AD Amsterdam,
The Netherlands. Tel: þ31 205665998; Fax: þ31 205669312; Email: [email protected]
Human Molecular Genetics, Vol. 13, No. 3 # Oxford University Press 2004; all rights reserved
296
Human Molecular Genetics, 2004, Vol. 13, No. 3
Table 1. Expression of genesa of the complement system in PNS and other tissues
Complement route
Tag
Gene
Classical
pathway
CTCTAAGAAG
C1Q, alpha
AATGAATGAA
AAATCAATAC
TTCTGTGCTG
ACTGAAGAA
AACACAGCCT
GGCCACGTAG
Alternative
pathway
Common
pathway
Regulatory
proteins
Receptors
Nerve (20 287)
Schwann cells (18 086)
8
0b
C1Q, beta
C1Q, gamma
C1R
C1S
C4
FD
1
3
5
3
2
16
GTTGTCTTTG
C3
CAACTAATTC
CLU
GGCTTGCTGA
CTTTCAAGA
TTGGGATGGG
CTCTCCAAAC
TGACTGGCAG
ATAGACATAA
ACTTTAATGA
DAF
MCP
HF
C1-INH
CD59
C1qBP
C5AR1
Brain (264 478)
Liver (66 861)
Fibroblasts (8 851)
1
3
0
1
0
1
1
0
0
<1c
0
0
<1
1
0
<1
<1
<1
5
20
<1
0
1
1
1
1
0
21
0
1
63
0
22
0
28
24
1
1
0
3
1
2
1
5
2
1
0
0
1
0
0
0
<1
0
1
8
1
<1
0
1
21
2
0
<1
<1
1
1
0
0
2
3
0
a
Expression per 10 000 tags. b0 means no tags present in analysed library. c<1 means expression lower than 1 tag per 10 000.
we show high representation of mRNA tags from genes
encoding genes of the complement system in a serial analysis
of gene expression (SAGE) library derived from adult human
sciatic nerve. The presence and localization of the encoded
proteins was analysed by western blot and immunohistochemistry. In addition, immunoreactivity directed to activated
complement components was found in neuroma samples, as
well as in rat sciatic nerve 4 h after crush injury. We propose
that the localized expression of regulatory complement factors
in the PNS is necessary for the protection of the nerve against
complement activation from outside and that the local
production of complement is essential for efficient myelin
clearance after nerve injury, a prerequisite for normal
regeneration and remyelination in the peripheral nerve.
RESULTS
High expression of complement components found
by serial analysis of gene expression
Our SAGE library from human adult sciatic nerve contained
9422 unique tags, of which 2279 tags (24.2%) were detected
more than once (from two to 264 times). Mapping the SAGE
tags to known genes and mRNAs in the GenBank database
showed expression of peripheral nerve-specific genes, a high
representation of genes involved in lipid metabolism as well as
housekeeping genes (22). Surprisingly, we also found a high
representation of components of the complement system.
Table 1 gives an overview of the various components of
complement found in the sciatic nerve SAGE library, in
comparison to a library constructed from cultured human
Schwann cells and three libraries obtained from the NCBI
SAGE data website (the Duke precrisis fibroblast library,
normal liver and a combination of all normal brain libraries are
given). Comparing the nerve and Schwann cell libraries
allowed us to identify genes specific for the total nerve
environment. Brain SAGE data were used to compare PNS and
CNS. The data from fibroblasts and liver were included because
they are a known source of complement.
In the nerve library, the classical pathway was represented by
C1QA, C1QB, C1QC, C1R, C1S and C4 (Table 2). From the
alternative pathway, the D component of complement (FD) was
highly represented. The central component C3 was highly
expressed. The regulatory components expressed in nerve
included clusterin (CLU ), decay accelerating factor (DAF ),
membrane cofactor protein (MCP), factor H (HF ), C1 inhibitor
(C1-INH ), CD59 and C1Q binding protein (C1QBP). The tag
from mRNA encoding the receptor C5AR1 was also present in
the nerve library. SAGE tags for C2, C5, C8, C9, PFC, Factor I
(IF ), C4BP, vitronectin, C1QR1, C3AR, CR1, CR2, Factor B
(FB) and properdin (PFC) were not present in the nerve library.
The high representation of the components of complement is
specific for the nerve environment, as the libraries constructed
from cultured fibroblasts and Schwann cells did not show high
expression of the complement components. The expression of
C3, FD and CLU was verified by northern blots and RT–PCR
analysis. The expression of C3, FD and CLU was verified by
northern blots and RT–PCR analysis using RNA from sciatic
nerve of five different individuals (data not shown).
Protein expression of complement components in sciatic
nerve, cortex and liver
Western blot analysis of samples from sciatic nerve, liver and
brain cortex was used to determine protein levels of the
complement components (Fig. 1). C1Q, C1R, C3, CLU, IF and
PFC could be detected in all three tissues. FD was only present
in the nerve. C1S, C5, C4BP and HF showed expression in
both liver and nerve. DAF, MCP, C3d and MAC antibodies
were not suitable for western blot analysis. None of the
complement proteins tested were detected in the Schwann cells
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þ
þþ
þþ
þ
ND
þ
ND
ND
/þ
/þ
þ
þ
þ
ND
þ
ND
ND
þ
þ
þ
þ
2
9
3
22
1
2
1
tags/10 000. bND not determined. () Negative staining; (þ) positive staining; (þþ) very positive staining.
a
þ
þ
9
5
3
16
21
C1Q
C1R
C1S
FD
Properdin
C3
C3c
C3d
C4
C5
C1-INH
C4BP
HF
IF
CLU
MCP
CD59
DAF
MAC-complex
þ
þ
þ
ND
ND
ND
þ
þ
þ
þ
þþ
þ
þ
þ
þ
þ
þ
þ
þ
þþ
þ
þþ
þ
þ
þ
NDb
ND
ND
þ
þ
þþ
þþ
þþ
þ
ND
þ
ND
ND
SC
To localize the site of protein expression in vivo we performed
immunohistochemistry on normal human nerve cross-sections.
The immunoreactivity of various anti-complement antibodies to
the different nerve components is summarized in Table 2 and
representative examples are given in Figure 2. Axons were
specifically immunostained by antibodies for C1S (Fig. 2A) and
C1R. CD59 antibody stained the myelin sheath (Fig. 2B). The
border of the myelin sheath, which probably contains cytoplasm
and nucleus of the Schwann cell, was stained with the antibodies
for C1S (Fig. 2A), C1R, C4BP (Fig. 2C), CLU (Fig. 2E) and DAF.
The endoneurium showed immunoreactivity to C1Q (Fig. 2D),
C1-INH, C3, CD59 and DAF, while C1R, C1S (Fig. 2A), C1INH, CD59 (Fig. 2B), C4BP (Fig. 2C), CLU (Fig. 2E) and DAF,
showed staining of the perineurium. The MAC was only detected
in the blood vessels of the nerve fibre (Fig. 2F). The erythrocytes
showed immunoreactivity to the C1Q antibody, C1-INH, CD59
and DAF. The FD antibody was not suitable for immunohistochemistry. The absence of staining with the antibodies CD68 and
leukocyte common antigen (LCA) confirmed the absence of
macrophages in these sections (data not shown).
þ
þ
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
ND
Endoneurium
Myelin
sheath
SC
Blood
vessels
Axon
Brain
Nerve
Myelin
sheath
Immunohistochemistry normal nerve
Western
Endoneurium
Epineurium
Axon
Protein localization by immunohistochemistry
SAGE
nervea
Liver
297
(data not shown). In all cases, the detected protein was of the
expected size.
Complement
factor
Table 2. Expression of complement factors in sciatic nerve on RNA and protein level
Immunohistochemistry neuromas
Epineurium
Blood
vessels
Human Molecular Genetics, 2004, Vol. 13, No. 3
Complement expression in disease
In order to see whether complement activation occurs in
diseases of the nerve, we analysed tissue from neurofibromatosis and neuromas. We tested for the presence of C3c and C3d,
breakdown products of C3b, a biologically active fragment of
C3 that is produced when complement is activated by either the
classical or alternative pathway. Deposition of these factors can
be used as an indicator of activation of the complement system.
Detailed analysis of six neuroma samples showed immunoreactivity for C3c and C3d (Table 2). The neuroma samples
showed staining of the perineurium of the sprouting fibres for
antibodies against the activated complement components, C3c
(Fig. 3A) and C3d, while normal human sciatic nerve showed
no staining (not shown). In other neuroma samples, proliferating Schwann cells reacted with antibodies against the activated
complement components (Fig. 3B and Table 2). In the neurofibromatosis sample the perineurium showed immunoreactivity
to both C3c (Fig. 3C) and C3d.
Complement expression after nerve injury
To study whether C-components also play a role in the initial
steps of Wallerian degeneration we performed nerve crush
experiments in rats. Several time points after nerve injury were
analysed. Already 4 h after nerve crush, severe damage of
the myelin sheath was seen. At the site of myelin damage C3c
was already detected (Fig. 3D) and remained present at 8 and
24 h. The reactivity was most pronounced at 4 and 8 h after
crush.
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Human Molecular Genetics, 2004, Vol. 13, No. 3
Figure 1. Expression of complement components in human cortex, liver and
sciatic nerve. Western blot analysis of protein extracts from human cortex, human
liver and human sciatic nerve protein using specific anti-complement antibody
fractions were performed as described in Materials and Methods. Equal amounts
of protein were loaded and confirmed by Coomassie staining (data not shown).
DISCUSSION
In this report, we show endogenous synthesis and expression of
components of the complement pathway in the normal human
peripheral nerve. The analysis of a gene expression profile
generated by SAGE allowed the identification of expression of
multiple components of the classical pathway (C1R, C1Q, C1S and
C4), of the alternative pathway (FD), and of the common pathway
(C3). Not only activating, but also inhibitory and regulatory proteins (CLU, C1-INH, C4BP, MCP, DAF and CD59) are expressed
in the peripheral nerve. We have confirmed the expression by
northern blot and RT–PCR analysis of mRNA extracted from
five different human sciatic nerve samples. The presence of the
complement proteins was demonstrated by western blot analysis.
The expression of complement by Schwann cells in the peripheral
nerve seems to depend on factors specific for the nerve environment, e.g. the differentiation state, as in vitro cultured fibroblasts
and Schwann cells do not show a high expression of the
components of the complement system. Immunohistochemistry
confirmed the presence of C1S, C1R, C3, C4BP, DAF, CD59 in
myelinating Schwann cells. The question whether all those complement components are produced by Schwann cells exclusively
cannot be answered by these experiments. The high levels of
mRNA for these proteins in the peripheral nerve suggest that
Schwann cells are the main source of mRNA synthesis. Thus, we
conclude that, like the CNS (3), the PNS has its own complement
biosynthesis. The results are in line with previous reports showing
the presence of the components of the complement system in the
rat and human sciatic nerve (23–26).
The finding that many complement genes are expressed in the
PNS is an example of the power of gene expression profiling.
Especially the use of SAGE or large microarrays covering a
substantial portion of, if not all known genes present in the
genome, allows datamining for pathways and co-regulated
genes. Since probes for a majority of the complement genes are
also present on large commercial microarrays, a study with these
arrays might also have identified these genes. However, SAGE
also allows identification of novel genes or transcripts.
The localization of the various complement components
differed considerably between axon, Schwann cell, endoneurium
and perineurium (Table 1). We propose that the regionalized
expression of the complement system might play a role in
regeneration of the PNS. In the normal nerve the first
components of the classical and alternative pathway were
present in the axon, but none of the regulatory components are
expressed, leaving the axon without direct protection. The
presence of CD59 protein in myelin protects the myelin sheath
from complement. This is in line with data from Koski et al.
(25), who described complement activation on myelin being
down-regulated at the step of the assembly of terminal
complement complexes, including C5b-9, due to the presence
of CD59. Vedeler et al. (27) showed that the presence of CR1
on the Schwann cell may be of importance in limiting damage
caused by the complement cascade. We did not find expression
of CR1 in the SAGE library of the nerve, but showed
expression of other inhibitory factors in the perineurium. The
scaffolding of the nerve, as well as the Schwann cell and
myelin, are thus protected from complement-induced damage
in the normal situation. We propose that, following disruption
of this architecture, rapid activation of the complement system
will take place. Shortly after nerve injury, Schwann cells
dedifferentiate, proliferate and actively initiate myelin degradation to facilitate nerve regeneration, a process called Wallerian
degeneration (28,29). Thus, activation of complement during
Wallerian degeneration can lead to rapid and efficient clearance
of the axons and subsequently myelin without damage to the
surrounding tissue. Previous research has shown that complement components affect both the ability of the macrophages to
invade the nerve and their ability to ingest myelin particles.
Bruck et al. (30) showed that during degeneration opsonization
of myelin is dependent on complement components, as
deficiency of C3 blocks myelin phagocytosis. Dailey et al.
(20) showed that after systemic depletion of C3 in Lewis rats
degeneration and regeneration after a crush injury of the sciatic
nerve was delayed and partially failed. Since complement
components might have multiple functions, depletion studies
cannot define the exact role of complement in nerve
degeneration and regeneration. One possibility is that proliferating Schwann cells activate complement to initiate myelin
degradation. To test this hypothesis, we have analysed two
disease states. First, we tested whether activated components of
complement (C3c and C3d) were present in chronic diseases
of the PNS, like neurofibromatosis and neuroma. Neuromas
occur in traumatized nerves in which the regeneration of
axons into the distal stump for some reason is made impossible.
A neuroma can best be considered as uncontrolled axonal
growth, supplemented by growth of Schwann cells, perineurial
cells, blood vessels, and connective tissue cells and fibres.
The presence of activated complement components in the
proliferating Schwann cells in the neuroma sample suggests
that Schwann cell are able to activate complement.
Human Molecular Genetics, 2004, Vol. 13, No. 3
299
Figure 2. Immunohistochemistry of complement components in the normal human sciatic nerve. Anti-C1S antibody stains axons and Schwann cells (A). AntiCD59-antibody stains the myelin sheath, endoneurium and epineurium (B). Anti-C4 binding protein antibody stains the Schwann cells, perineurium and blood
vessels (C). Anti-C1q antibody stains erythrocytes and endoneurium (D). Anti-CLU antibody stains perineurium and Schwann cells (E). Anti-MAC antibody stains
only the blood vessels in the nerve (F). Scale bar is 100 mm.
Furthermore, we studied nerve degeneration induced in rats
by nerve crush injury. Already 4 h after nerve crush,
immunoreactivity towards activated complement components
was found. Immunoreactivity was seen in the myelin sheath of
the injured nerve (Fig. 3). This is in line with previous findings.
Hays et al. (11) showed the presence of C3d on the myelin
sheath surface of patients with immune-mediated neuropathies
and suggested that on the surface of the Schwann cell a
mechanism must exist which induces degradation of C3b into
C3c and C3d. Combining our results with those of Hays et al.
suggests that the proliferating Schwann cells themselves might
be able to activate complement. A role of complement
activation in GBS has also recently been suggested by
Wanschitz et al. (31). They found C9neo deposition on
degenerating myelin sheets in acute cases of GBS.
In summary, our data provides evidence for the presence of
an endogenous biosynthesis of many components of the
complement system in the sciatic nerve. The presence of
activated components of the complement system after acute
and chronic nerve injury suggests an active role for the
complement system in peripheral nerve regeneration. In
addition, Reichert and Rothshenker (32) showed that complement components participate in myelin phagocytosis and
therefore activation of endogenous complement in the PNS
might play an important role in the remodeling of the PNS.
We propose that the local biosynthesis of complement contributes to protection of the nerve, possibly by facilitating the
maintenance, repair and regeneration of peripheral nerve myelin.
MATERIALS AND METHODS
Tissue and RNA extraction
Sciatic nerve samples were obtained at routine autopsy from
patients who had died of heart failure without prior history of
peripheral nerve disease. Autopsies were performed within 12 h
after death. Tissue was immediately frozen in liquid nitrogen.
The tissue was homogenized using a mikro-dismembrator (B.
Braun Biotech International, Melsungen, Germany). RNA was
directly extracted from the tissue using Trizol (LifeTechnologies,
Gaithersburg, MD, USA). Human peripheral nerve neuromas
were obtained from patients with chronic neuroma pain after
traumatic peripheral nerve lesions. The neuroma samples as well
as the neurofibromatosis samples were retrieved from the tissue
bank present in the Academic Medical Centre, Amsterdam, The
Netherlands. Informed consent was obtained from each patient
according to the hospital standards.
Construction of SAGE library
The SAGE library was constructed from 1 mg of sciatic nerve
poly-A-RNA, derived from one individual, following SAGE
Protocol 1.0c by Velculescu et al. (33) (www.sagenet.org).
Data were analysed using USAGE V2 software developed
in our institute (34) for extraction of single tags from
sequence data and subsequent identification on the EMBL
human gene database. To further study tag identification and
300
Human Molecular Genetics, 2004, Vol. 13, No. 3
Figure 3. Activated complement components C3c is present in chronic and acute disease of the peripheral nerve. C3C is detected in neuroma, neurofibromatosis
and after crush injury. Neuroma: perineurium of the sprouting nerve fibres (A) and the proliferating Schwann cells (B) show reactivity to the anti-C3c antibody.
Neurofibromatosis: C3c is present in the perineurium (C). Crush injury: immunoreactivity to the C3c antibody is observed 4 h after nerve injury at the site of injury
(D). Scale bar is 100 mm.
expression, NCBI/CGAP’s SAGEMAP program was used
(www.ncbi.nlm.nih.gov/SAGE).
RNA analysis
Northern blots were prepared from nerve samples from five
different individuals, as well as from cultured human Schwann
cells and human brain cortex. ten micrograms of total RNA
were glyoxilated and size separated on a 1% agarose gel,
prepared using the glyoxal/NaPi electrophoresis method (35).
Capillary blotting onto a nylon filter (N-Hybond, Amersham,
UK) was performed overnight in 20 SSC, followed by ultraviolet cross-linking (0.2 J/cm2) and baking (80 C/2 h).
Hybridizations and post-hybridization washes were according
to the protocols of Church and Gilbert (35). (RT–PCR) was
performed to obtain probes for the C3, CLU and D components
of complement (primer sequences are available upon request).
Hybridized probe was visualized and quantified with a Fuji
BAS 1800 Imager (Fuji, Raytest Benelux B.V., Tilburg, The
Netherlands) and analysed with AIDA software Raytest
(Raytest Benelux B.V).
Quantitative PCR
Real-time RT–PCR on the LightCycler (Roche Diagnostics,
Mannheim, Germany) was performed in a total volume of
10 ml; 10 reaction buffer (Taq polymerase, dNTPs, SYBR
Green, Roche Diagnostics), 4 mM MgCl2, 20 nM of each
oligonucleotide and cDNA or water as negative control were
added. Reactions were subjected to an initial denaturation step
of 30 s at 95 C, followed by 45 cycles of 10 s at 95 C, 5 s at the
specific annealing temperature (GAPDH, 60 C; C3 and CLU,
55 C) and 10 s 72 C. At the end of each cycle, the fluorescence
emitted by the DNA bound SYBR Green was measured. After
completion of the cycling process, samples were subjected to a
temperature ramp (from 5 C above annealing temperature to
95 C at 2 C/s) with continuous fluorescence monitoring for
melting curve analysis. Apart from primer-dimers, a single
narrow peak was obtained for each PCR product by melting
curve analysis at the specific melting temperature, and only a
single band of the predicted size was observed on agarose gel
electrophoresis. Expression levels were normalized to the
expression of GAPDH. All experiments were performed in
triplicate.
Western blot analysis
Human cerebral cortex, liver and nerve were homogenized
using a micro-dismembrator in liquid nitrogen. The homogenates were resuspended in 20 mM Tris–HCl, 6% glycerol,
0.4% SDS and 5 mM DTT. Protein extracts were boiled for
5 min, separated by SDS–PAGE using 10% polyacrylamide
gels, and transferred to nitrocellulose filters. The nitrocellulose filters were pre-incubated in 50 mM Tris–HCl buffered
saline containing 0.5% Tween-20 (TBST) and 5% non-fat dry
milk powder. Blots were incubated for 3 h with the primary
antibody (Table 3) in TBST containing 5% non-fat dry
milk. Membranes were washed in TBST and incubated with
horseradish peroxidase-conjugated secondary antibody for 2 h.
Membranes were washed in TBST and immuno-reactivebands
Human Molecular Genetics, 2004, Vol. 13, No. 3
Table 3. Antibodies used for western blot and immunohistochemistry
301
Nerve crush injury
Antiserum
Source
Host species
Dilution
western
Dilution
immuno
C1q
C1r
C1s
C1-INH
C3
C3d
C3c
CR3
MAC
C4BP
MCP
DAF
CD59
CLU
FD
HF
CD-68
LCA
IF
PFC
Nordic
Biodesign
Nordic
Sigma
Cappel
DAKO
DAKO
DAKO
Sigma
Calbiochem
Quidel
Quidel
Gifta
Quidel
Giftb
Quidel
Dako
Dako
Quidel
Quidel
Rabbit
Rabbit
Goat
Rabbit
Goat
Rabbit
Rabbit
Mouse
Mouse
Rabbit
Mouse
Mouse
Mouse
Mouse
Rabbit
Goat
Mouse
Mouse
Goat
Goat
1 : 1000
1 : 1000
1 : 1000
1 : 2000
1 : 3000
ND
ND
1 : 2000
ND
1 : 1000
ND
ND
1 : 1000
1 : 1000
1 : 200
1 : 2000
ND
ND
1 : 2000
1 : 2000
1 : 250
1 : 250
1 : 250
1 : 250
1 : 2000
1 : 1000
1 : 1000
ND
1 : 100
1 : 1000
1 : 50
1 : 100
1 : 800
1 : 1000
ND
ND
1 : 100
1 : 200
ND
ND
a
Kindly provided by S. Asghar, Department of Dermatology, AMC, The
Netherlands. bKindly provided by R. Veerhuis, Department Pathology, VU, The
Netherlands. ND ¼ not determined.
were detected using enhanced chemiluminescence (ECL,
Amersham, Piscataway, NJ, USA).
Immunohistochemistry
Frozen nerve sections (5 mm) were fixed on glass slides with
acetone. Endogenous peroxidase was inactivated by a 30 min
incubation in 0.3% H2O2 in PBS. Immunohistochemical
staining was performed by a three-step immunoperoxidase
technique. The slides were incubated with normal goat serum
for 10 min and then incubated with the first antibody diluted in
BSA for 60 min (Table 3), followed by incubation for 30 min
with a 1 : 300 dilution of a biotinylated secondary antibody
in PBS/10% human AB serum (DAKO). They were then
incubated for 30 min with horseradish peroxidase labelled
polystreptavidin (ABC-complex, DAKO). Peroxidase activity
was visualized by incubation of the slides with 0.05% 3-amino9-ethylcarbazole in acetate buffer for 10 min followed by a
counterstaining with hematoxylin for 30 s. Parafin-embedded
nerve sections (7 mm) of the neuroma, neurofibromatosis and
rat sciatic nerve samples were deparafined using xylol and an
ethanol sequence. Endogenous peroxidase activity was inactivated by a 30 min incubation in 0.3% H2O2 in methanol. Slides
were heated at full power in a microwave in citrate buffer
0.01 M pH 6.0 for 3 min. The immunohistochemical staining
was performed as described above. All incubations were
performed at room temperature. Slides incubated with
secondary antibody or the isotype alone served as negative
controls. Antibody dilutions were determined on skin biopsy
samples from complement positive psoriasis patients and
healthy controls. Images were captured using a digital camera
(Colorview12) and analysis software (AnalySIS, Soft Imaging
Systems GmbH, Zoeterwoude, The Netherlands).
Twelve-week-old PVG rats weighing 150–200 g (Harlan, UK)
were housed in pairs in plastic cages in the animal house and
given rat chow and water ad libitum. All the surgical
procedures were performed with aseptic techniques. For nerve
crush, the animals were anaesthetized by interaperitoneal
injection of a mixture of ketamin (Eurovet, The Netherlands),
rompun (Bayer, Germany) and atropine (Eurovet, The
Netherlands) in a ratio of 4 : 2 : 1. The right sciatic nerve was
exposed through a gluteal muscle splitting incision. At this
location, the nerve trunk was crushed for 30 s period between
an artery clamp and a stitch was placed at the site of the crush.
On the left side, a control operation was performed which
exposed the sciatic nerve but did not disturb it, and a stitch was
also placed. The muscle and skin were then closed with
stitches. At each selected post-operative time (0, 4, 8, 12, 18
and 24 h), two rats were anaesthetized and intracardially
perfused with 10% formaldehyde. Both sciatic nerves were
removed and each nerve was divided into six pieces. The nerve
pieces were placed in formaldehyde for post sampling fixation
overnight and then processed and embedded in paraffin. The
blocks were sectioned serially at 7 mm. Sections were stained
with an antibody against C3c, as described above. Luxol fast
blue staining was performed to determine the quality of the
sample and the morphological changes due to the nerve crush.
ACKNOWLEDGEMENTS
This work was supported by a grant from MDA, USA.
We thank R. Veerhuis and N. Okada for kindly providing the
antibodies. We thank M. Ramkema for the help with the
immunostaining and A. Meintjes for the help with the RNA
analysis. We thank Drs A.L.M.A. ten Asbroek, S.S. Asghar,
A. Rozemuller, P. Eikelenboom, L. Kalaydjieva and
J.M.B.V. de Jong for their support, encouragement and critical
reading of the manuscript.
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