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Human Molecular Genetics, 2004, Vol. 13, No. 3 295-302
DOI: 10.1093/hmg/ddh029

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,*

1Neurogenetics 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


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 membrane-embedded 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, 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
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


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  		Table 1. Expression of genesa of the complement system in PNS and other tissues
 
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).


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  		Table 2. Expression of complement factors in sciatic nerve on RNA and protein level
 
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 (data not shown). In all cases, the detected protein was of the expected size.



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  		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).

 
Protein localization by immunohistochemistry
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), C1-INH, 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).



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  		Figure 2. Immunohistochemistry of complement components in the normal human sciatic nerve. Anti-C1S antibody stains axons and Schwann cells (A). Anti-CD59-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 µm.

 
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.



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  		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 µm.

 
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.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
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.

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
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
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 µg 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 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 20xSSC, followed by ultra-violet 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 µl; 10x 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 were detected using enhanced chemiluminescence (ECL, Amersham, Piscataway, NJ, USA).


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  		Table 3. Antibodies used for western blot and immunohistochemistry
 
Immunohistochemistry
Frozen nerve sections (5 µm) 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-amino-9-ethylcarbazole in acetate buffer for 10 min followed by a counterstaining with hematoxylin for 30 s. Parafin-embedded nerve sections (7 µm) 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).

Nerve crush injury
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 µm. 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.


   FOOTNOTES
 
* To whom correspondence should be addressed at: Neurogenetics Laboratory, Academic Medical Center, K2-213, PO Box 22660, 1100 AD Amsterdam, The Netherlands. Tel: +31 205665998; Fax: +31 205669312; Email: f.baas{at}amc.uva.nl


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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