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Molecular Immunology 46 (2009) 523–531

Contents lists available at ScienceDirect

Molecular Immunology

journa l homepage: www.e lsev ier .com/ locate /mol imm

vidence for a novel chemotactic C1q domain-containing factor in the leecherve cord

uriel Tahtouha, Francoise Croqa, Jacopo Vizioli a, Pierre-Eric Sautierea, Christelle Van Campa,ichel Salzeta, Mohamed R. Dahab, Joël Pestela,∗,1, Christophe Lefebvrea,1

Université de Lille 1 (USTL), Laboratoire de Neuroimmunologie des Annélides, Centre National de la Recherche Scientifique, FRE 2933,FR 147, F59655 Villeneuve d’Ascq, FranceDepartment of Nephrology, Leiden University Medical Center, Postbox 9600, Leiden 2300 RC, The Netherlands

r t i c l e i n f o

rticle history:eceived 23 June 2008eceived in revised form 15 July 2008ccepted 15 July 2008vailable online 25 October 2008

eywords:1qhemotaxisicroglial cells

a b s t r a c t

In vertebrates, central nervous system (CNS) protection is dependent on many immune cells includingmicroglial cells. Indeed, activated microglial cells are involved in neuroinflammation mechanisms by inter-acting with numerous immune factors. Unlike vertebrates, some lophotrochozoan invertebrates can fullyrepair their CNS following injury. In the medicinal leech Hirudo medicinalis, the recruitment of microglialcells at the lesion site is essential for sprouting of injured axons. Interestingly, a new molecule homologousto vertebrate C1q was characterized in leech, named HmC1q (for H. medicinalis) and detected in neuronsand glial cells. In chemotaxis assays, leech microglial cells were demonstrated to respond to human C1q.The chemotactic activity was reduced when microglia was preincubated with signaling pathway inhibitors(Pertussis Toxin or wortmannin) or anti-human gC1qR antibody suggesting the involvement of gC1qR inC1q-mediated migration in leech. Assays using cells preincubated with NO chelator (cPTIO) showed that

eech

erve cord repairnnate immunity

C1q-mediated migration was associated to NO production. Of interest, by using anti-HmC1q antibodies,HmC1q released in the culture medium was shown to exhibit a similar chemotactic effect on microglialcells as human C1q.

In summary, we have identified, for the first time, a molecule homologous to mammalian C1q inleech CNS. Its chemoattractant activity on microglia highlights a new investigation field leading to better

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. Introduction

In vertebrates, the microglial cell is considered as one ofhe most important cells involved in the immune protection ofhe central nervous system (CNS). Following CNS injury, theseuiescent cells are activated by numerous inflammatory stimuliNakamura et al., 1999; Piehl and Lidman, 2001), become amoe-oid and are recruited to the lesion sites where they participate

n the elimination of cellular debris and apoptotic bodies (Aloisi,001; Hanisch, 2002). Activated microglial cells are involved in

euroinflammation mechanisms by synthesizing a large panel ofro-inflammatory mediators (Nakamura, 2002; Qin et al., 2006)nd numerous factors that control the innate immune responseKielian, 2004).

∗ Corresponding author. Université Lille1, CNRS Fre 2933, Bâtiment SN3.el.: +33 320 335 942; fax: +33 320 434 054.

E-mail address: joel.pestel@univ-lille1.fr (J. Pestel).1 The authors supervised equally this work.

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161-5890/$ – see front matter © 2008 Elsevier Ltd. All rights reserved.oi:10.1016/j.molimm.2008.07.026

echanisms.© 2008 Elsevier Ltd. All rights reserved.

Among the mediators expressed by microglial cells and neurons,he subunit C1q belonging to C1 complement factor seems to be aey-molecule in neuroinflammatory diseases, i.e. systemic lupusrythematosus (Flierman and Daha, 2007; Trendelenburg, 2005).ndeed, C1q was described to opsonise apoptotic cells for clearancey phagocytes (Fishelson et al., 2001; Nauta et al., 2002; Trouwt al., 2008). In the absence of such opsonization (e.g. in C1q or4 deficiency), apoptotic cells may remain longer in the body andtimulate autoantibody production or undergo pro-inflammatoryecondary necrosis (Botto, 1998, 2001; Botto and Walport, 2002).tudies in C1q-deficient mice have shown the presence of a largeumber of apoptotic bodies in CNS (Botto, 1998, 2001; Botto andalport, 2002). C1q was also involved in various neurodegen-

rative pathologies as Alzheimer disease (Bergamaschini et al.,001; Tacnet-Delorme et al., 2001). Following exposure to the amy-

oid beta peptide, microglial cells exhibit chemotactic migration inrder to remove the peptide, but this interaction might be inhib-ted by C1q (Webster et al., 2000). Thus in vertebrate CNS, innatemmune interactions between C1q and microglial cells may be cru-ial in the regulation of neuroinflammation dependent diseases.

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n addition, chemotactic properties of C1q were demonstrated forendritic cells. Of interest, their migration is mediated throughecognition of different types of C1q binding proteins (Vegh et al.,006).

Unlike vertebrates, some lophotrochozoan invertebrates canully repair their CNS following injury (experimental crush or sec-ion). The medicinal leech Hirudo medicinalis is a recognized modeln neuroscience. Indeed, previous studies reported that the medic-nal leech CNS can be efficiently regenerated (Baylor and Nicholls,971; Jansen and Nicholls, 1972).

During this repair, a microglial cell accumulation at the lesionite is observed (Morgese et al., 1983; von Bernhardi and Muller,995), as similarly described in mammals. Recent studies in leechtrongly suggest that microglial cells are essential for the usualprouting of injured axons (Ngu et al., 2007). In order to char-cterize potential immune factors involved in the recruitment ofhe microglial cell population, analyses of Expressed Sequenceag (EST) library established from leech CNS were carried out.

molecule having significant homology with vertebrate C1qas been characterized. In the present study, we identified a H.edicinalis C1q domain containing (C1qDC) factor named HmC1q

Genbank Accession number, EU581715). Interestingly, we demon-trated its implication in the recruitment of leech microglial cellsollowing experimental injury by using in vitro chemotaxis assays.

. Materials and methods

.1. Leech central nervous system structure

The medicinal leech H. medicinalis is well studied in neurobiol-gy because the CNS structure is tightly defined (Coggeshall andawcett, 1964). Leech CNS is included in a blood sinus and is con-

ig. 1. Diagram of ganglion from leech CNS. The dorsal view of the ganglion showsour of the six packet glial cells enveloping neuron cell bodies. The axonal processesass through the neuropil and are prolonged into connectives. The neuropil liesorso-medially and has two macroglial cells. Microglial cells are distributed in gan-lia and connectives. The nervous system is enclosed in the outer capsule whichs covered on the outside by a visceral layer of the endothelium (lining the ventrallood sinus).

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nology 46 (2009) 523–531

tituted by 32 metameric ganglia connected by connectives. Fig. 1chematically represents a dorsal view of typical midbody gan-lion which contains around 400 neuron cell bodies separated in 6roups each one enveloped by a packet glial cell. Their axonal pro-esses and neuritic trees pass through the neuropil containing twoacroglial cells (Coggeshall and Fawcett, 1964). Leech CNS containsicroglial cells distributed all along the nerve cord. Their number

s about 10 000–14 000 for each ganglion and about 2000 for eachonnective (Morgese et al., 1983). The endothelial layer present athe surface of the nerve cord constitutes an important exchangerea with blood of the surrounding sinus.

.2. Leech CNS preparation

H. medicinalis adult leeches were obtained from RicarimpexEysines, France). After anesthesia in 10% ethanol at 4 ◦C for 15 min,nimal CNS (nerve cords) were dissected out in a sterile Ringer solu-ion (115 mM NaCl, 1.8 mM CaCl2, 4 mM KCl, 10 mM Tris maleate,H 7.4) under a laminar flow hood. After isolation, samples werelaced in 3 successive baths of antibiotics (100 UI/ml penicillin,00 �g/ml streptomycin and 100 �g/ml gentamycin) for 15 min andurther incubated in Leibovitz L-15 medium (Gibco, Invitrogen)omplemented with 2 mM l-glutamin, 0.6% glucose and 10 mMepes (complete medium). The experimental injury was performedy crushing or cutting one of the two connectives between thirdnd fourth ganglia. Then, in situ hybridization and immunohisto-hemical analyses were performed at several time points (T = 0 h or= 6 h).

.3. Microglial cell preparation

Nerve cords were placed in 35 mm Petri dishes with 200 �l ofomplete L-15 medium. Each ganglion was carefully decapsulatedy removing the collagen layer enveloping the nerve cord withicroscissors. Nerve cells, neurons (10–70 �m) and microglial cells

5 �m), were mechanically resuspended by gentle scraping andltered through a 7 �m nylon mesh. The enriched microglial cellopulation was then collected according to the size and centrifugedt 1000 × g for 10 min at room temperature (RT). The cell pelletas resuspended in complete medium (100 �l per nerve cord) forigration assays.

.4. Conditioned medium (CM) preparation

Eight nerve cords which have been crushed or cut were cul-ured during 24 h as described above. The medium (500 �l) washen centrifuged 20 min at 1000 × g to eliminate cells and tissues.he supernatant was finally used as conditioned medium (CM) forestern blot and chemotaxis experiments.

.5. Molecular characterization

Partial cDNA sequence of interest was completed by 5′ RACECR. H. medicinalis total RNA were prepared to construct aACE cDNA library using BD SMARTTM RACE cDNA Amplificationit following manufacturer’s protocol (Clontech). Reverse tran-cription was performed using antisense primers deduced fromST sequence (C1q-Rv 5′-TCAAAACGTCCTCATCCATCGTCA-3′). PCRmplifications were performed with an elongation time of 5 min.

elected PCR products were ligated into PGEM T-easy vector andloned into JM109 cells according to the manufacturer’s instruc-ions (Promega). Finally, products were sequenced using the BigDyeerminator v3.0 polymerization kit before detection on the ABIrism 310 Genetic Analyzer (Applied Biosystems). BLAST programs

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ere used for sequence analysis (Altschul et al., 1997; Karlin andltschul, 1990).

.6. Fluorescent in situ hybridization (FISH)

Nerve cords were fixed for an hour at 4 ◦C in 4% paraformalde-yde just after dissection or 6 h after incubation in complete L-15edium. 5’ biotin labeled specific antisense probes and sense probe

negative control) were generated from a linearized cDNA plasmidy in vitro transcription using DIG/Biotin RNA-labeling kit accordingo the manufacturer’s instructions (Roche). The hybridization pro-ocol was followed as previously described (Nardelli-Haefliger andhankland, 1992). Nerve cords were incubated with a secondarynti-biotin antibody conjugated to Alexa Fluor 488 (Invitrogen)iluted 1:5000 in PBS, rinsed with PBS and finally mounted withlycergel (Sigma Life Science). Samples were kept at 4 ◦C in the darkntil observation with a Zeiss LSM 510 Laser Scanning Confocalicroscope.

.7. Western blotting

CNS protein extract analysis was performed from 10 nerveords as previously described (Vergote et al., 2006). Twenty-fiveicrograms of proteins were loaded onto SDS-PAGE. Immunoblot

nalyses were also carried out using purified conditioned mediums follow. CM (500 �l) was acidified with 0.1% Trifluoroaceticcid (TFA) and proteins were prepurified on SepPak C18 cartridge

Waters). Elution was performed with 50% acidified acetonitrile0.1% TFA). The fraction was then dried and reconstituted in 20 �lf Læmmli buffer before loading onto the SDS-PAGE. It was per-ormed according to Tastet and collaborators with a 12% acrylamideunning gel and a 4% acrylamide stacking gel (Tastet et al., 2003).igration was performed using a cathode buffer (0.6% Tris base,

.5% Taurine, and 0.1% SDS) and an anode buffer (0.6% Tris base,

.8% Glycine, and 0.1% SDS). Gels ran at 70 V for 15 min and at20 V for 45 min. Separated proteins were transferred to Nitrocel-ulose Transfer Membrane Protran® BA 83 (Schleicher & Schuell) bylectroblotting. After preincubation in blocking solution (BS) (PBSontaining 0.05% Tween 20 and 2% ovalbumin fraction V) mem-ranes were incubated overnight at 4 ◦C with specific antisera:ouse monoclonal anti-human gC1qR (Santa Cruz Biotechnolo-

ies), rabbit polyclonal anti-HmC1q or pre-immune serum (dilution:1000 in BS). Specific rabbit polyclonal anti-HmC1q antibodiesere raised using a synthetic peptide corresponding to predictedis197-Thr212 region of HmC1q protein (Agrobio). After three PBSashes, goat anti-rabbit or anti-mouse IgG antibodies conju-

ated with horseradish peroxidase (dilution 1:20 000) (Jacksonmmunoresearch) were added for 1 h at RT. The final washes wereerformed in PBS and immunolabelled proteins were revealed withhe ECL Kit SuperSignal West Pico Chemoluminescent SubstratePierce) and Kodak® X-Omat LS film (Sigma–Aldrich).

.8. Immunohistochemistry

In experiments with anti-human C1q antibody, analyses wereerformed on dissociated nerve cells (see above), cytocentrifugedShandon Cytospin® 3) at RT for 8 min at 2000 × g on poly-l-lysine-oated slides and stored at 4 ◦C. Nerve cords or dissociated nerveells were fixed for 60 min at 4 ◦C in 4% paraformaldehyde just afterissection or 6 h after incubation in complete L-15 medium. Fixed

amples were washed in PBS, permeabilized by a 24 h-incubationt RT in 1% Triton X100 in PBS and preincubated for 8 h at RTn 1% Triton, 3% Normal Donkey Serum (NDS) and 1% Ovalbuminn PBS. Samples were then incubated overnight at 4 ◦C with rab-it polyclonal anti-HmC1q antibodies (1:5000) or rabbit polyclonal

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nology 46 (2009) 523–531 525

nti-human C1q antibodies diluted in PBS containing 1% BSA, 0.05%riton, 1% NDS and 1% ovalbumin. After three washes with PBS, don-ey anti-rabbit IgG antibody (Invitrogen) conjugated to Alexa Fluor88 was added. Secondary antibodies incubation, sample mountingnd confocal microscope observation were performed as for FISHxperiments. Samples without the addition of primary antibodyere used as negative control.

.9. Chemotaxis assay

Chemotaxis assays were performed by using the double-P assays described by Köhidai et al. with minor modifications (Kohidai,995). Thirty-five millimeters Petri dishes were filled with 1 ml of a.5% agar and 1% gelatin solution. After drying, two 6 mm diameterells were done, each one presenting a parallel individual channel.ne well was filled with 50 �l of purified microglial cells and

he other one with chemotactic factors or negative controls. Ahannel was further created perpendicularly to others with aoverslip. One hour later, cells in the chemoattractant containingell were collected. Either human C1q (0, 5, 25, 50 and 100 nM) or

onditioned medium (50 �l) were used as chemotactic molecules.n any experiment, negative controls were carried out with com-lete L-15 medium alone. Inhibitory chemotactic experimentsere performed with neutralizing antibodies and scavengers.oncerning the study of human C1q, cells were preincubatedor 30 min at RT with mouse monoclonal anti-human gC1qRntibody (dilution 1:25), Pertussis Toxin (PTX) (500 ng/ml), G-rotein inhibitors: wortmannin (10 mM) or nitric oxide chelator:-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-xide (cPTIO, 50 �M) (Sigma–Aldrich). Concerning the study ofeech-conditioned medium, cells were preincubated for 30 mint RT with rabbit polyclonal anti-HmC1q antibody (dilutions:100; 1:50; 1:25) and, as negative control, pre-immune serumdilution 1:25). The number of migrating cells was counted on

hemocytometer (three different counts) under Zeiss Axioskopicroscope. Experiments were done in triplicate. The results were

xpressed as the mean cell number ± S.D. Comparisons betweeneans were made using Student’s t-test. Statistical differencesere considered to be significant if p was <0.01.

. Results

.1. Molecular characterization of HmC1q

EST library was constructed from total RNA of nerve cordssolated from adult leeches. Following sequencing and identifica-ion analyses, molecules homologous to complement factors weredentified. We focused our attention on a C1q domain containing

olecule. In order to achieve its complete nucleotide sequence,′RACE-PCRs based on specific EST library cDNA were carriedut. Final molecular cloning has led to identify 1837 nucleotidesequence presenting an open reading frame (960 nucleotides) cod-ng a 320-amino acids protein (Fig. 2A). The analysis of the sequencey Signal-P V1.1 program predicted a putative signal peptide corre-ponding to amino acid regions 1–23 of the protein (Nielsen et al.,997). The putative mature chain (24–320) has a predicted molec-lar weight of 32.6 kDa. Translated sequence has been compared

n databases with the BLAST-P and BLAST-X programs (Altschul etl., 1997; Karlin and Altschul, 1990). Interestingly, the sequence

ontains two conserved domains usually related to vertebrate1q domain containing (C1qDC) protein family: the collagen-likeomain containing several G–X–Y motifs and the globular C1qomain (Fig. 2A). In addition, BLAST-P alignment program showedignificant homologies with mammalian C1qDC proteins. Thus,

526 M. Tahtouh et al. / Molecular Immunology 46 (2009) 523–531

Fig. 2. Molecular characterization of HmC1q molecule in leech nerve cord. (A) Molecular characterization of HmC1q reveals a full-length cDNA containing a coding region of960 bases (nucleotide positions 106–1066). The amino acid sequence (320 amino acids), which was deduced from this nucleotide sequence is constituted of a putative signalpeptide (highlighted in light grey, 1–23), a collagen-like region (highlighted in light grey, 102–158) which contains G–X–Y repeat domains (G are highlighted in black) and theC1q domain (highlighted in dark grey, 173–312). Numbers for nucleotides and amino acids are, respectively, indicated on left and right of the sequence. (B) Multiple alignment

M. Tahtouh et al. / Molecular Immunology 46 (2009) 523–531 527

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e named the molecule as HmC1q (Genbank accession numberU581715) according to the conserved domains and homologies.

Since HmC1q sequence presents similarities with many ver-ebrate C1qDC proteins, we preferred to limit the comparison touman molecules (Corpet, 1988) (Fig. 2B). Relative homologies inmino acid sequences with leech C1q, indicated in the legend ofig. 2, vary between 41% and 45%. Similar homology values werebtained for C1q families of other mammals.

Finally, western blot analysis using rabbit polyclonal anti-mC1q antibody allowed detection of a product (about 33 kDa),hich is relevant with the mature form (residues 24–320) of known1q proteins (Fig. 2C). Pre-immune serum was used as negativeontrol. In addition, because the analysis used protein extract fromervous system culture medium, the presence of specific signal

ndicates that HmC1q protein may be released by nervous cell pop-lations during in vitro repair processes.

.2. HmC1q mRNA distribution in the leech nervous system

In order to locate the presence of related HmC1q transcripts inhe leech nervous system, specific fluorescence in situ hybridiza-

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rograms show two conserved domains which are the collagen domain and the C1q domaiepresented in grey (relatively conserved) and black (highly conserved). In order to presen1QTNF5 (Accession Q9BXJ0), C1QTNF1 (Accession Q9BXJ1), C1QA (Accession P02745), C1QTNF7 (Accession Q9BXJ2), C1QTNF6 (Accession Q9BXI9), ADIPOQ (adiponectin) (Acc5, 43, 45, 45, 44, 44 and 41% homologies with HmC1q. (C) Western blotting analysis waserum as negative control (lane 1), rabbit polyclonal anti-HmC1q antibody revealed a pro

y images show mRNA localization using antisense HmC1q riboprobes immediatelywing injury in a packet of neurons (C). Sense probes were used as negative controls0 �m.

ions were carried out on whole nerve cords (Fig. 3). The transcriptsre constitutively present in neuron cell bodies outside of theucleus and mainly at the periphery of the cytoplasm. Fluores-ent probes were also detected in some nerve fibers of connectiveselying metameric ganglia (Fig. 3A). Six hours after nerve injury,ranscripts were only observed in ganglion compartments includ-ng neuron bodies and packet glial cells (Fig. 3C). Similar results

ere obtained in whole ganglia (Fig. 3C, inset) and dissociated neu-ons (data not shown). No specific signals were detected with senserobes (Fig. 3B and D).

.3. Immunolocalization of HmC1q protein

Distribution of HmC1q was showed in different elements ofeech nervous system after injury. By using polyclonal anti-human1q antibodies with isolated leech neurons, the staining was lim-

ted to cell body cytoplasm (Fig. 4A). A similar pattern was observedn neurons stained with specific anti-HmC1q polyclonal antibodies.ndeed, the anti-HmC1q signal was clearly detected on the con-ocal microscopy image of one packet of neurons (Fig. 4B). Theanglion endothelium layer and the packet glial cell surround-

n (respectively underlined with single line and double line). Conserved residues aret large molecular diversity, the alignment does only consider the human molecules.1QB (Accession P02746), C1QC (Accession P02747), C1QTNF9 (Accession Q5VX65),ession Q15848) and C1QTNF8 (Accession P60827) respectively present 46, 45, 40,realized from conditioned medium protein extract. Comparatively to pre-immuneduct of about 33 kDa (lane 2).

528 M. Tahtouh et al. / Molecular Immunology 46 (2009) 523–531

Fig. 4. Fluorescent immunostaining of leech CNS by using anti-C1q antibodies. (A) Isolated neurons are immunopositive with anti-human C1q antibody. (B–G) Confocalmicroscope analysis of the immunostaining obtained with rabbit pre-immune serum or anti-leech C1q (anti-HmC1q) antibodies. (B) Detail of stained neurons from wholeg unopod of staiw ale ba

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anglion. (C) Optical section through a packet of neurons. Arrowheads show immetected on samples treated with pre-immune serum as negative control. (E) Detailas respectively observed immediately (T = 0 h) and 6 h (T = 6 h) following injury. Sc

ng neuron cell bodies were stained (Fig. 4C) with a massiveccumulation of HmC1q in the apical region of that macroglialell (data not shown). A similar sample, treated with rabbit pre-mmune serum as a negative control did not show any signalFig. 4D). At the connective level, the end of axonal processes

site of lesion) (Fig. 4E) and the endothelial cells (Fig. 4F and) were also immunopositive for HmC1q. Interestingly, following

njury, the protein appeared as spread at the surface of the tissueFig. 4F) and, 6 h later, was mainly associated to endothelial cellsFig. 4G).

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sitive signal for the macroglial cell enveloping neuron bodies. (D) No signal wasned cut end of injured connectives (site of lesion). (F and G) Endothelial cell stainingrs correspond to 20 �m.

.4. Chemotactic effects of human C1q and leech C1q (HmC1q) oneech microglial cells

When leech nervous system is injured, a microglial cell migra-ion towards the lesion site is observed (Morgese et al., 1983). In

ertebrates, some complement factors were reported to contributeo the chemotaxis of cells involved in inflammatory responses.ecause HmC1q factor presents sequence homologies with human1q, the leech microglia recruitment was evaluated using purifieduman C1q. Freshly prepared leech microglial cells were found

M. Tahtouh et al. / Molecular Immunology 46 (2009) 523–531 529

Fig. 5. (A) Chemotactic effect of human C1q on leech microglia migration. Microglialcells were tested against human C1q gradient (0, 5, 25, 50 and 100 nM) in order tostudy the chemotactic property and determine the optimal concentration of humanC1q in chemotaxis. (B) Inhibitory effects on human C1q-mediated chemotaxis onleech microglia. Preincubations of microglial cells with cPTIO (50 �M), anti-humangC1qR antibody (1:25), wortmannin (10 mM) and PTX (500 ng/ml) were performedto analyze the mechanisms of microglia accumulation. Human C1q (25 nM) was usedas chemoattractant molecule in all experimental sets and replaced by complete L-15 medium in negative controls. (C) Western blot analysis was performed from CNSprotein extract using a mouse monoclonal gC1qR antibody. Signal showed numerousproducts ranged from 45 to 75 kDa. Chemotaxis results were realized from threeindependent experiments and expressed in number of migrating cells (as the meancell number ± S.D.). (A) Asterisks denote that cell migration of the indicated samplew(o

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Fig. 6. Chemotactic effect of conditioned medium on leech microglia. Conditionedmedium was used in all experimental sets and replaced by complete L-15 mediumin negative controls. Microglial cells were preincubated with different dilutions(serum:water, 1:50, 1:25) of anti-HmC1q antibodies or pre-immune serum (1:25) ascontrol. The results from three independent experiments were expressed in num-bmo

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as significantly different (p < 0.01) than the previous concentration of human C1q0 vs. 5; 5 vs. 25; 25 vs. 50; 50 vs. 100 nM). (B) Asterisks denote that cell migrationf the indicated sample was significantly different (p < 0.01) than all other ones.

o migrate towards a human C1q concentration gradient in aose-dependent manner with a maximal migration at 25 nMFig. 5A). Higher concentrations of human C1q produced a dimin-shed microglia migration corroborating the known chemotacticroperties of most molecules in chemotaxis assays. The optimaligration with 25 nM human C1q was used as positive control

n following experiments. As previously described, nitric oxideNO) was reported to interfere in the regulation of leech microglia

ccumulation (Chen et al., 2000; Kumar et al., 2001). First, wevaluated the influence of NO chelator (cPTIO) on C1q-dependenthemotaxis. The results showed that cPTIO preincubated withicroglial cells significantly reduced cell migration towards

uman C1q compared to positive control (Fig. 5B). In parallel, other

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xperiments were undertaken to specify the signaling pathwaynvolved in C1q-mediated chemotaxis. Thus, two inhibitors weresed: the pertussis toxin (PTX) as a inactivator of G protein andhe wortmannin as a PtdIns(3,4,5)P3 kinase inhibitor. Using PTX,

icroglial cell migration towards the optimal C1q concentrationas strongly reduced (Fig. 5B). In addition, wortmannin appeared

o be a potent inhibitor of C1q-mediated chemotaxis (Fig. 5B). Bothortmannin and PTX at the tested concentrations were not known

o be toxic to the microglial cells, as assessed by the shape andhe number of non-migrating cells. Finally, inhibition of microglialell migration was investigated by using specific anti-humanC1qR antibody, known to recognize part of the receptor whichinds to the globular domain of the C1q molecule. Addition of thisntibody at the same time with the deposit of microglial cell ledo a significant reduction of the chemotactic response inducedith the optimal dose of human C1q (Fig. 5B). Of interest, the

nti-gC1qR antibody enabled the detection of different productsanged from 45 to 75 kDa in the CNS protein extract (Fig. 5C).

Otherwise, the conditioned medium (CM) obtained from aeech nervous system culture after a 24 h incubation exhibited

chemotactic effect similar to that observed with human C1qFig. 6). Incubation of microglial cells with different concentrationsf the specific anti-HmC1q antibody induced a significant dose-ependent decrease of cell recruitment which indirectly illustrateshe importance of HmC1q in leech microglia accumulation (Fig. 6).ndeed, anti-HmC1q antibody did specifically recognize HmC1q inM (Fig. 2C). Moreover, CM-dependent migration was not modi-ed when cells were incubated with pre-immune serum (Fig. 6).hus, results demonstrated the specific implication of HmC1q inell recruitment and corroborated the response of leech microgliao purified human C1q.

. Discussion

The C1q family proteins are considered as having an ancient

volutionary history (Endo et al., 2006). A molecule homologous toammalian C1q was recently characterized in lamprey (Matsushita

t al., 2004). By taking into account its capacity to bind GlcNAc,amprey C1q was considered as a lectin and as an initial recogni-ion molecule of the complement system in innate immunity before

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30 M. Tahtouh et al. / Molecular

he establishment of adaptive immunity (Matsushita et al., 2004).n deuterostome invertebrates, such a molecule was also foundn sea urchin and can opsonise pathogens (Melillo et al., 2006).nterestingly, the history seems to be older. Our results empha-ize the presence of C1q related factor in protostome invertebrates.ndeed, for the first time, this report shows the existence of a C1qomain-containing protein, named HmC1q, in the nervous sys-em of lophotrochozoans, the leech H. medicinalis and suggests itsnvolvement in early mechanisms during nervous system repair.he existence of C1q-like molecules has never been reported innvertebrate nervous system to date. The sequence analysis ofmC1q revealed high similarities with vertebrate C1q proteins buto significant homologies has been observed with invertebrates. As

t exists for HmC1q, the C1q family is characterized by a C-terminalonserved globular C1q domain (gC1q), which is structurally sim-lar to the TNF domain and a collagen domain. Notably, HmC1qontains residues in gC1q domain which are conserved in all knownC1q containing proteins (Kishore et al., 2004).

In vertebrates, members of C1q family, the C1qTNF proteinsre involved in triggering and regulation of various inflammatoryeactions (Kishore et al., 2004). In most cases, their globular car-oxyl terminal portion recognizes broad categories of moleculese.g. PAMPs) and the collagen-like N-terminal domain links thenvading organism to complement mediated or phagocyte effec-or mechanisms of the immune system. Reports mentioned that1qTNF1 factor might have a promising anti-platelet thromboticctivity and most likely by pacifying the thrombogenic site ofascular injury (Lasser et al., 2006). Their key-role in neuroin-ammatory mechanisms was also underlined. Indeed, C1qTNF5

actor is implicated in late-onset retinal degeneration (Shu et al.,006). In many neurodegenerative diseases, the globular head of1q is involved in the capture of apoptotic cells and consequently

n the regulation of the local inflammatory reaction. Recent experi-ents demonstrated that C1q can control neurogenesis process by

agging unwanted synapses in order to favor their complement-ependent elimination. This physiological mechanism might beberrantly reactivated in some neurodegenerative diseases. Thus,n the mouse glaucoma model, neuronal C1q, which is normallyown-regulated in the adult CNS, might be early up-regulated andynaptically relocalized in the adult retina during the glaucoma dis-ase (Stevens et al., 2007). Finally some C1q domain containingroteins belonging to cerebellin subfamily expressed at the level ofNS synapses were recently considered as transneuronal cytokinesYuzaki, 2008).

As previously described, unlike in mammals, leech neuronsurvive to lesion injury and regrow processes to restore the con-ections between two ganglia. Thus, we investigated the role ofmC1q factor in leech CNS repair following injury. Interestingly,

mmediately after injury, HmC1q mRNA were observed in someeuron cell bodies of segmental ganglia and in some nerve fibersaxonal processes) of connectives. HmC1q protein was present ineuron cell bodies but no specific signal was detected in axonal pro-esses. In whole ganglion, immunopositive signals were observedn packet glial cells and endothelial layer. Confocal immunochem-stry details of the endothelium showed that HmC1q protein waspread at the surface of the tissue. Six hours post-injury, whereasmC1q transcripts were only observed in some neuron cell bodies,stronger signal was detected for the protein equally distributed

n the endothelium area. From these observations, we hypothesizehat, following injury, HmC1q mRNA might be immediately trans-

ated in axonal processes and proteins could be quickly secreted.ndeed, the protein is rapidly accumulated at the cut end of connec-ives and later massively present in packet glial cells and endothelialrea as a potential release process towards the blood sinus. Thus,he fast HmC1q protein release at the cut ends of axons might

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nology 46 (2009) 523–531

romote the recruitment of microglial cells that we have furthernalyzed. The presence of HmC1q at the endothelium level sur-ounding the nerve cord is of interest. Indeed, the endothelial layerhows some irregularities and large numbers of small invaginationshich allow active transport between nervous system and blood

Coggeshall and Fawcett, 1964). In a rat model of arterial hyperten-ion, the induction of C1q expression was reported in glomerularndothelium as a potential repair mechanism of the capillary wallamage (Kreutz et al., 2007). Similarly, in leech, HmC1q might beonfined to the endothelial cells as well as to the lesion site enablingn efficient nerve cord repair. Further studies will investigate theotential localization of HmC1q protein in the blood sinus following

njury.The chemotactic activity of C1q was then evaluated. Indeed, in

eech, one of the early steps of response to nerve cord injury ishe recruitment of microglial cells at lesions. In our experimental

odel, we clearly observed that leech microglial cells responded indose-dependent manner to human C1q as well as to conditionededium containing characterized HmC1q factor. The chemotac-

ic activity of human C1q on immune cells was already reportednd may implicate gC1qR and G-protein signaling (Leigh et al.,998). The fact that human C1q exhibited such activities on inver-ebrate cells might be related to the sequence homology observedetween leech and human molecules and mainly to the pres-nce of the C1q domain which was described as conservativeotifs along evolution. Of interest, western blot analysis from leech

erve cord culture medium using anti-HmC1q antibody showedhe presence secreted HmC1q. In order to determine the path-ay by which HmC1q factor might act, signaling inhibitors were

ncubated with leech microglia. The first important point relatedo these inhibitory experiments was the demonstration that thelobular part of the C1q molecule plays a key-role for an efficientinding to microglial cells and potentially induces efficient sig-als. Indeed, C1q-mediated chemotaxis did appear to be sensitiveo PTX treatment and specific inhibitors of PtdIns3 kinase such asortmannin. The results strongly suggested the potential involve-ent of G-proteins and PtdIns3 kinase in propagating the C1q

nduced signal for chemotaxis. In vertebrates, interaction of C1qith gC1qR exhibited similar signal pathways (Leigh et al., 1998). In

ddition, immature dendritic cells that express C1q receptors wereeported to respond in dose-dependent manner to soluble C1q ando migrate to inflammatory sites (Vegh et al., 2003, 2006). In leech,y considering the inhibitory effect observed when microglial cellsere incubated with anti-human gC1qR antibody, we can hypothe-

ize that microglial cells might express similar receptor for HmC1q.his hypothesis is supported by western blot analysis. Indeed, thepecificity of monoclonal anti-gC1qR antibody enabled the detec-ion of products whose size is relevant with known multimerizedC1qR forms (Jha et al., 2002; Lim et al., 1996). In addition, theharacterization of such a potential receptor from nervous sys-em EST is underway. The second important point is the using ofnitric oxide scavenger (cPTIO). The report showed the effect ofO chelation on microglia migration in human C1q gradient. Thehemotactic effect is not completely abolished, probably becausef partial inhibition of NO. In any case, the results bring evidencehat the C1q effect is related to the presence of NO which plays aey-role in leech microglia recruitment required for efficient CNSepair (Chen et al., 2000; Kumar et al., 2001; Ngu et al., 2007). Fur-her studies will investigate the interactions between production ofO and production of leech C1q (HmC1q). Experiments using the

mC1q recombinant form and anti-HmC1q antibodies will enable

o specify its chemotactic activity and characterize the potentialpecific receptors on target cells. In conclusion, the original evi-ence of HmC1q in leech CNS highlights a new investigation fieldo better understand nervous repair mechanisms.

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vfrom lesions in leeches. J. Neurobiol. 27, 353–366.

M. Tahtouh et al. / Molecular

cknowledgements

Authors would like to thank Dr. Christian Slomianny and Pr.atalia Prevarskaya (Inserm, U800, Laboratoire de Physiologie Cel-

ulaire, Université de Lille 1, France) for access to Zeiss LSM 510aser Scanning Confocal Microscope; Gilles Courtand (Centre Com-un de Mesures Imagerie Cellulaire, Université de Lille 1, France)

nd Annie Desmons, Mathilde Verstraete and Céline Wichlacz forechnical assistance. Supported by grants from Centre Nationale la Recherche Scientifique (CNRS) and Ministère de L’Educationationale, de L’Enseignement Supérieur et de la Recherche (France).. medicinalis nervous system EST databases were granted byirudinea Genomic Consortium.

eferences

loisi, F., 2001. Immune function of microglia. Glia 36, 165–179.ltschul, S.F., Madden, T.L., Schaffer, A.A., Zhang, J., Zhang, Z., Miller, W., Lipman, D.J.,

1997. Gapped BLAST and PSI-BLAST: a new generation of protein database searchprograms. Nucleic Acids Res. 25, 3389–3402.

aylor, D.A., Nicholls, J.G., 1971. Patterns of regeneration between individual nervecells in the central nervous system of the leech. Nature 232, 268–270.

ergamaschini, L., Donarini, C., Gobbo, G., Parnetti, L., Gallai, V., 2001. Activation ofcomplement and contact system in Alzheimer’s disease. Mech. Ageing Dev. 122,1971–1983.

otto, M., 1998. C1q knock-out mice for the study of complement deficiency inautoimmune disease. Exp. Clin. Immunogenet. 15, 231–234.

otto, M., 2001. Links between complement deficiency and apoptosis. Arthritis Res.3, 207–210.

otto, M., Walport, M.J., 2002. C1q, autoimmunity and apoptosis. Immunobiology205, 395–406.

hen, A., Kumar, S.M., Sahley, C.L., Muller, K.J., 2000. Nitric oxide influences injury-induced microglial migration and accumulation in the leech CNS. J. Neurosci. 20,1036–1043.

oggeshall, R.E., Fawcett, D.W., 1964. The fine structure of the central nervous systemof the leech, Hirudo medicinalis. J. Neurophysiol. 27, 229–289.

orpet, F., 1988. Multiple sequence alignment with hierarchical clustering. NucleicAcids Res. 16, 10881–10890.

ndo, Y., Takahashi, M., Fujita, T., 2006. Lectin complement system and patternrecognition. Immunobiology 211, 283–293.

ishelson, Z., Attali, G., Mevorach, D., 2001. Complement and apoptosis. Mol.Immunol. 38, 207–219.

lierman, R., Daha, M.R., 2007. Pathogenic role of anti-C1q autoantibodies in thedevelopment of lupus nephritis—a hypothesis. Mol. Immunol. 44, 133–138.

anisch, U.K., 2002. Microglia as a source and target of cytokines. Glia 40, 140–155.ansen, J.K., Nicholls, J.G., 1972. Regeneration and changes in synaptic connections

between individual nerve cells in the central nervous system of the leech. Proc.Natl. Acad. Sci. U.S.A. 69, 636–639.

ha, B.K., Salunke, D.M., Datta, K., 2002. Disulfide bond formation through Cys186facilitates functionally relevant dimerization of trimeric hyaluronan-bindingprotein 1 (HABP1)/p32/gC1qR. Eur. J. Biochem. 269, 298–306.

arlin, S., Altschul, S.F., 1990. Methods for assessing the statistical significance ofmolecular sequence features by using general scoring schemes. Proc. Natl. Acad.Sci. U.S.A. 87, 2264–2268.

ielian, T., 2004. Microglia and chemokines in infectious diseases of the nervoussystem: views and reviews. Front Biosci. 9, 732–750.

ishore, U., Gaboriaud, C., Waters, P., Shrive, A.K., Greenhough, T.J., Reid, K.B., Sim,R.B., Arlaud, G.J., 2004. C1q and tumor necrosis factor superfamily: modularityand versatility. Trends Immunol. 25, 551–561.

ohidai, L., 1995. Method for determination of chemoattraction in Tetrahymena pyri-formis. Curr. Microbiol. 30, 251–253.

reutz, R., Schulz, A., Sietmann, A., Stoll, M., Daha, M.R., de Heer, E., Wehland, M.,2007. Induction of C1q expression in glomerular endothelium in a rat modelwith arterial hypertension and albuminuria. J. Hypertens. 25, 2308–2316.

umar, S.M., Porterfield, D.M., Muller, K.J., Smith, P.J., Sahley, C.L., 2001. Nerve injury

induces a rapid efflux of nitric oxide (NO) detected with a novel NO microsensor.J. Neurosci. 21, 215–220.

asser, G., Guchhait, P., Ellsworth, J.L., Sheppard, P., Lewis, K., Bishop, P., Cruz, M.A.,Lopez, J.A., Fruebis, J., 2006. C1qTNF-related protein-1 (CTRP-1): a vascular wallprotein that inhibits collagen-induced platelet aggregation by blocking VWFbinding to collagen. Blood 107, 423–430.

W

Y

nology 46 (2009) 523–531 531

eigh, L.E., Ghebrehiwet, B., Perera, T.P., Bird, I.N., Strong, P., Kishore, U., Reid, K.B.,Eggleton, P., 1998. C1q-mediated chemotaxis by human neutrophils: involve-ment of gClqR and G-protein signalling mechanisms. Biochem. J. 330 (Part 1),247–254.

im, B.L., Reid, K.B., Ghebrehiwet, B., Peerschke, E.I., Leigh, L.A., Preissner, K.T., 1996.The binding protein for globular heads of complement C1q, gC1qR. Functionalexpression and characterization as a novel vitronectin binding factor. J. Biol.Chem. 271, 26739–26744.

atsushita, M., Matsushita, A., Endo, Y., Nakata, M., Kojima, N., Mizuochi, T., Fujita,T., 2004. Origin of the classical complement pathway: lamprey orthologue ofmammalian C1q acts as a lectin. Proc. Natl. Acad. Sci. U.S.A. 101, 10127–10131.

elillo, D., Sfyroera, G., De Santis, R., Graziano, R., Marino, R., Lambris, J.D., Pinto, M.R.,2006. First identification of a chemotactic receptor in an invertebrate species:structural and functional characterization of Ciona intestinalis C3a receptor. J.Immunol. 177, 4132–4140.

orgese, V.J., Elliott, E.J., Muller, K.J., 1983. Microglial movement to sites of nervelesion in the leech CNS. Brain Res. 272, 166–170.

akamura, Y., 2002. Regulating factors for microglial activation. Biol. Pharm. Bull.25, 945–953.

akamura, Y., Si, Q.S., Kataoka, K., 1999. Lipopolysaccharide-induced microglial acti-vation in culture: temporal profiles of morphological change and release ofcytokines and nitric oxide. Neurosci. Res. 35, 95–100.

ardelli-Haefliger, D., Shankland, M., 1992. Lox2, a putative leech segment identitygene, is expressed in the same segmental domain in different stem cell lineages.Development 116, 697–710.

auta, A.J., Trouw, L.A., Daha, M.R., Tijsma, O., Nieuwland, R., Schwaeble, W.J., Gin-gras, A.R., Mantovani, A., Hack, E.C., Roos, A., 2002. Direct binding of C1q toapoptotic cells and cell blebs induces complement activation. Eur. J. Immunol.32, 1726–1736.

gu, E.M., Sahley, C.L., Muller, K.J., 2007. Reduced axon sprouting after treatmentthat diminishes microglia accumulation at lesions in the leech CNS. J. Comp.Neurol. 503, 101–109.

ielsen, H., Engelbrecht, J., Brunak, S., von Heijne, G., 1997. A neural network methodfor identification of prokaryotic and eukaryotic signal peptides and predictionof their cleavage sites. Int. J. Neural. Syst. 8, 581–599.

iehl, F., Lidman, O., 2001. Neuroinflammation in the rat—CNS cells and their role inthe regulation of immune reactions. Immunol. Rev. 184, 212–225.

in, B., Cartier, L., Dubois-Dauphin, M., Li, B., Serrander, L., Krause, K.H., 2006. Akey role for the microglial NADPH oxidase in APP-dependent killing of neurons.Neurobiol. Aging 27, 1577–1587.

hu, X., Tulloch, B., Lennon, A., Vlachantoni, D., Zhou, X., Hayward, C., Wright, A.F.,2006. Disease mechanisms in late-onset retinal macular degeneration associ-ated with mutation in C1QTNF5. Hum. Mol. Genet. 15, 1680–1689.

tevens, B., Allen, N.J., Vazquez, L.E., Howell, G.R., Christopherson, K.S., Nouri, N.,Micheva, K.D., Mehalow, A.K., Huberman, A.D., Stafford, B., Sher, A., Litke, A.M.,Lambris, J.D., Smith, S.J., John, S.W., Barres, B.A., 2007. The classical complementcascade mediates CNS synapse elimination. Cell 131, 1164–1178.

acnet-Delorme, P., Chevallier, S., Arlaud, G.J., 2001. Beta-amyloid fibrils activatethe C1 complex of complement under physiological conditions: evidence fora binding site for A beta on the C1q globular regions. J. Immunol. 167, 6374–6381.

astet, C., Lescuyer, P., Diemer, H., Luche, S., van Dorsselaer, A., Rabilloud, T., 2003.A versatile electrophoresis system for the analysis of high- and low-molecular-weight proteins. Electrophoresis 24, 1787–1794.

rendelenburg, M., 2005. Antibodies against C1q in patients with systemic lupuserythematosus. Springer Semin. Immunopathol. 27, 276–285.

rouw, L.A., Blom, A.M., Gasque, P., 2008. Role of complement and complementregulators in the removal of apoptotic cells. Mol. Immunol. 45, 1199–1207.

egh, Z., Goyarts, E.C., Rozengarten, K., Mazumder, A., Ghebrehiwet, B., 2003.Maturation-dependent expression of C1q-binding proteins on the cell surfaceof human monocyte-derived dendritic cells. Int. Immunopharmacol. 3, 345–357.

egh, Z., Kew, R.R., Gruber, B.L., Ghebrehiwet, B., 2006. Chemotaxis of humanmonocyte-derived dendritic cells to complement component C1q is mediatedby the receptors gC1qR and cC1qR. Mol. Immunol. 43, 1402–1407.

ergote, D., Macagno, E.R., Salzet, M., Sautiere, P.E., 2006. Proteome modificationsof the medicinal leech nervous system under bacterial challenge. Proteomics 6,4817–4825.

on Bernhardi, R., Muller, K.J., 1995. Repair of the central nervous system: lessons

ebster, S.D., Yang, A.J., Margol, L., Garzon-Rodriguez, W., Glabe, C.G., Tenner, A.J.,2000. Complement component C1q modulates the phagocytosis of Abeta bymicroglia. Exp. Neurol. 161, 127–138.

uzaki, M., 2008. Cbln and C1q family proteins—new transneuronal cytokines. CellMol. Life Sci..