microglial cells constitute the resident immune cells , maintaining the integrity of the nervous system by constantly surveying the environment . under pathological conditions , they rapidly change and turn to amoeboid activated microglia in order to migrate over long distances and accumulate at lesions . the microglia in the leech hirudo medicinalis were the first cells to be named  microglia  by del rio - hortega using his silver carbonate method   and are considered as the first microglia model . leech microglial cells represent the only cell population capable of moving through the nerve cord . otherwise , they can not be mistaken with other glial cells that have a larger size and a different shape . because individual sensory cells regenerate new synaptic connections with a high degree of specificity after a section , the involvement of microglia was investigated in that natural nerve repair process . the leech nerve cord is tubular and is not vascularized within the blood sinus . in this context , by using an isolated and injured segment of nerve cord maintained in tissue culture , some authors reported that synapse regeneration is possible following the accumulation of resident microglia at the crush site and without any blood cell . even in the presence of blood sinus , a very low infiltration of blood cells is observed in injured cns , which highlights the importance of the resident microglia at the lesions . their accumulation has been shown to be essential for the usual sprouting of injured axons . the mobility of activated resident microglia has been associated with morphological changes from stellate to rounded shape , comparable to those of activated microglia in mammals , and is dependent on specific chemotactic signals [ 10,1416 ] . among these chemotactic factors , we demonstrated the role of hmc1q ( for hirudo medicinalis ) in the microglial cell accumulation after leech cns injury . in mammals , it is well established that c1q recognizes the c1qr(p ) receptor to contribute to the phagocytosis of debris by microglia . some authors showed the importance of c1q in the microglial activation   and a neuroprotective role for c1q in early inflammatory response , but the c1q - dependent recruitment of mammalian microglia at lesions is still poorly understood . the chemotactic properties of c1q have been shown towards blood immune cells [ 2224 ] and some authors specified that such chemotactic activity occurs through the recognition of both gc1qr ( alias c1qbp ) and calreticulin ( also known as cc1qr , calr , or crt ) receptors on peripheral dendritic cells . in the leech cns , hmc1q contributes to the microglial recruitment by interacting with a recently characterized receptor , named hmc1qbp , which is homologous to mammalian gc1qr . the present report shows the involvement of a second receptor in the c1q - dependent microglial activation . this report establishes that the presence of hmcalr in the leech microglia represents a part of the c1q - dependent reactivity . h. medicinalis adult leeches were obtained from ricarimpex ( eysines , france ) . the leech nerve cord ( cns ) is constituted by the head ganglion , 21 body ganglia , and 7 fused tail ganglia . the ganglia are joined by structures , called connectives , containing the axonal processes and glial cells . after anesthesia in 10% ethanol at 4c for 15 min , animal cns were dissected out in a sterile ringer solution ( 115 mm nacl , 1.8 mm cacl2 , 4 mm kcl , 10 mm tris maleate , ph 7.4 ) under a laminar flow hood . after isolation , samples were placed in 3 successive baths of antibiotics ( 10 ui / ml penicillin , 10 g / ml streptomycin , and 10 g / ml gentamicin ) for 15 min and further incubated in leibovitz l-15 medium ( invitrogen , usa ) containing 2 mm l - glutamin , 0.6% glucose , and 10 mm hepes ( complete medium ) . the experimental injury was performed by crushing the connectives between the third and fourth ganglia of an isolated fragment . nerve cords were used for fluorescence in situ hybridization , whole mount immunohistochemistry , or nerve cell preparation . for microglial cell isolations , nerve cords treated as indicated above were placed in 35-mm petri dishes with 200 l of complete l-15 medium . each ganglion was carefully decapsulated by removing , with micro - scissors , the collagen layer enveloping the nerve cord . nerve cells , neurons , and microglial cells were mechanically dissociated by gentle scraping ( total nerve cells ) . after a filtration through 7-m nylon mesh as described , the enriched microglial cell population was then collected and centrifuged at 1000  g for 10 min at rt . the cell pellet was resuspended in l-15 medium ( 100 l per nerve cord ) for migration assays . the analysis of hirudo medicinalis genome allowed in silico prediction of mrna databases according to intro - exon boundaries . based on the candidate sequence detected , forward and reverse primers were designed to frame the complete sequence of predicted mrna . from total rna extracted from leech nerve cord using trizol reagent and according to the manufacturer s procedure ( invitrogen , usa ) the calreticulin - related molecule was amplified by pcr using the specific forward ( 5ggtagcaatacgtgcagtttg3 ) and reverse ( 5gcaaccaagagtaggcaacc3 ) primers and the platinum 
 taq dna polymerase according to the manufacturer s instructions ( invitrogen , usa ) . selected pcr products were ligated into pgem t - easy vector and cloned into jm109 cells according to the manufacturer s instructions ( promega , usa ) . finally , products were sequenced using bigdye terminator v3.0 polymerization kit before detection on genetic analyzer ( applied biosystems , usa ) . blast programs were used for sequence analysis in databases and comparison with initial predicted mrna sequence . nerve cords were fixed for one hour at 4c in 4% paraformaldehyde just after dissection . the 5 biotin - labeled specific antisense probe and sense probe ( negative control ) were generated from the 8421479 nucleotides sequence of hmcalr mrna ( genbank accession number kf709537 ) . after pcr amplification and the insertion of the product in pgem - t easy vector system ( promega , usa ) , the rna sequence of interest was obtained by in vitro transcription using dig / biotin rna - labeling kit according to the manufacturer s instructions ( roche , switzerland ) . nerve cords were incubated with a secondary anti - biotin antibody conjugated to alexa fluor 488 ( dilution 1 : 5000 in pbs ) ( invitrogen , usa ) . samples were rinsed with pbs . prior to mounting with glycergel ( sigma life science , usa ) , the cell nuclei were counterstained by hoechst 33342 fluorescent dye ( 1:1,000 , invitrogen usa ) for 10 min . slides were kept at 4c in the dark until observation with a zeiss lsm700 confocal microscope . in experiments with anti - human calreticulin antibody , analyses were performed on nerve cords dissected out as described above . they were fixed for 1 hour at 4c  immediately after dissection ( t=0 ) or 24 h ( t24h ) after incubation in complete l-15 medium  in 4% paraformaldehyde , washed in pbs , permeabilized by a 24-h incubation at rt in 1% triton x100 in pbs and pre - incubated for 8 h at rt in 1% triton , 3% normal donkey serum ( nds ) and 1% ovalbumin in pbs . samples were then incubated overnight at 4c with specific rabbit polyclonal anti - human calreticulin antibodies ( santa cruz biotechnology , usa ) diluted in a pbs solution ( 1:250 ) containing 1% bsa , 0.05% triton , 1% nds , and 1% ovalbumin . the anti - calreticulin antibodies were directed against an antigen sequence corresponding to the lys - leu region of human calreticulin , presenting 81% homology with leech calreticulin . after 3 washes with pbs , samples were incubated 1 h at room temperature with anti - rabbit donkey antibody ( invitrogen , usa ) conjugated to alexa fluor 488 ( 1:2000 ) in a pbs solution containing 1% bsa , prior to mounting , the cell nuclei were counterstained by hoechst 33342 fluorescent dye ( 1 : 1000 , invitrogen usa ) for 10 min . the biotinylation of the recombinant human c1q ( prospecbio , usa ) was carried out by using the sulfo - nhs - ss - biotin kit ( pierce , usa ) according to the manufacturer s instructions . unreacted sulfo - nhs - ss - biotin was removed using the zeba desalt spin columns ( pierce , usa ) . biotinylated human c1q was immediately fixed onto a streptavidin column ( pierce , usa ) , previously equilibrated with 5 volumes of pbs 0.1 m . microglia protein extract ( 800 g ) was added in the column , incubated overnight at 4c , and rinsed 10 times with pbs 0.1 m . captured microglial cell proteins were eluted from the streptavidin - agarose with 5% 2-mercaptoethanol - pbs 0.1 m at 30c for 30 min . proteins were precipitated in 10% trichloroacetic acid / acetone at 20c for 45 min and centrifuged at 13000  g for 15 min . the protein pellet was washed in cold acetone , air dried and dissolved in lmmli buffer . two other columns were used for the negative controls : the first containing the biotinylated human c1q without any microglia protein extract , and the second containing only the microglia protein extract to evaluate the unspecific reaction between streptavidin and microglial cell components . the membrane was incubated for 30 min at rt in blocking solution ( 0.05% tween , milk powder 5% w / v in pbs ) and then overnight at 4c in rabbit polyclonal anti - human calreticulin antibodies ( santa cruz biotechnology , usa ) , diluted at 1:5000 in blocking solution containing 1% ovalbumin . after rinsing with pbs-0.05% tween , the membrane was incubated for 1 h at rt in secondary goat anti - rabbit polyclonal antibodies conjugated with horseradish peroxidase ( jackson immunoresearch , usa ) ( dilution 1:20000 ) . the signal was revealed using the ecl kit supersignal west dura chemoluminescent substrate ( pierce , usa ) on kodak x - omat ls film ( sigma - aldrich , usa ) . in vitro chemotaxis assays were performed by using the double - p assay as described by khidai et al . with minor modifications . thirty - five millimeter petri dishes were filled with 1 ml of a 0.5% agar and 1% gelatin solution . after drying , one well was filled with 50 l of purified microglial cells ( see above ) and the other with chemotactic factor or negative controls reagents . either vehicle ( non - transformed pischia pastoris culture supernatant ) or recombinant hmc1q ( rhmc1q ) produced in p. pastoris   alone or with soluble calr were used ( 8 l ) as chemotactic factors . for inhibitory chemotactic experiments , cells were pre - incubated for 1 h at rt either with rabbit polyclonal anti - human calreticulin antibodies ( 1:1000 ) or with rabbit igg isotype as negative control ( 1:1000 ) . the number of migrating cells was counted on a hemocytometer ( 3 different counts ) under an axioskop microscope ( zeiss , germany ) . h. medicinalis adult leeches were obtained from ricarimpex ( eysines , france ) . the leech nerve cord ( cns ) is constituted by the head ganglion , 21 body ganglia , and 7 fused tail ganglia . the ganglia are joined by structures , called connectives , containing the axonal processes and glial cells . after anesthesia in 10% ethanol at 4c for 15 min , animal cns were dissected out in a sterile ringer solution ( 115 mm nacl , 1.8 mm cacl2 , 4 mm kcl , 10 mm tris maleate , ph 7.4 ) under a laminar flow hood . after isolation , samples were placed in 3 successive baths of antibiotics ( 10 ui / ml penicillin , 10 g / ml streptomycin , and 10 g / ml gentamicin ) for 15 min and further incubated in leibovitz l-15 medium ( invitrogen , usa ) containing 2 mm l - glutamin , 0.6% glucose , and 10 mm hepes ( complete medium ) . the experimental injury was performed by crushing the connectives between the third and fourth ganglia of an isolated fragment . nerve cords were used for fluorescence in situ hybridization , whole mount immunohistochemistry , or nerve cell preparation . for microglial cell isolations , nerve cords treated as indicated above were placed in 35-mm petri dishes with 200 l of complete l-15 medium . each ganglion was carefully decapsulated by removing , with micro - scissors , the collagen layer enveloping the nerve cord . nerve cells , neurons , and microglial cells were mechanically dissociated by gentle scraping ( total nerve cells ) . after a filtration through 7-m nylon mesh as described , the enriched microglial cell population was then collected and centrifuged at 1000  g for 10 min at rt . the cell pellet was resuspended in l-15 medium ( 100 l per nerve cord ) for migration assays . the analysis of hirudo medicinalis genome allowed in silico prediction of mrna databases according to intro - exon boundaries . based on the candidate sequence detected , forward and reverse primers were designed to frame the complete sequence of predicted mrna . from total rna extracted from leech nerve cord using trizol reagent and according to the manufacturer s procedure ( invitrogen , usa ) , cdna were synthesized using an oligo(dt ) priming . the calreticulin - related molecule was amplified by pcr using the specific forward ( 5ggtagcaatacgtgcagtttg3 ) and reverse ( 5gcaaccaagagtaggcaacc3 ) primers and the platinum 
 taq dna polymerase according to the manufacturer s instructions ( invitrogen , usa ) . selected pcr products were ligated into pgem t - easy vector and cloned into jm109 cells according to the manufacturer s instructions ( promega , usa ) . finally , products were sequenced using bigdye terminator v3.0 polymerization kit before detection on genetic analyzer ( applied biosystems , usa ) . blast programs were used for sequence analysis in databases and comparison with initial predicted mrna sequence . nerve cords were fixed for one hour at 4c in 4% paraformaldehyde just after dissection . the 5 biotin - labeled specific antisense probe and sense probe ( negative control ) were generated from the 8421479 nucleotides sequence of hmcalr mrna ( genbank accession number kf709537 ) . after pcr amplification and the insertion of the product in pgem - t easy vector system ( promega , usa ) , the rna sequence of interest was obtained by in vitro transcription using dig / biotin rna - labeling kit according to the manufacturer s instructions ( roche , switzerland ) . nerve cords were incubated with a secondary anti - biotin antibody conjugated to alexa fluor 488 ( dilution 1 : 5000 in pbs ) ( invitrogen , usa ) . samples were rinsed with pbs . prior to mounting with glycergel ( sigma life science , usa ) , the cell nuclei were counterstained by hoechst 33342 fluorescent dye ( 1:1,000 , invitrogen usa ) for 10 min . slides were kept at 4c in the dark until observation with a zeiss lsm700 confocal microscope . in experiments with anti - human calreticulin antibody , analyses were performed on nerve cords dissected out as described above . they were fixed for 1 hour at 4c  immediately after dissection ( t=0 ) or 24 h ( t24h ) after incubation in complete l-15 medium  in 4% paraformaldehyde , washed in pbs , permeabilized by a 24-h incubation at rt in 1% triton x100 in pbs and pre - incubated for 8 h at rt in 1% triton , 3% normal donkey serum ( nds ) and 1% ovalbumin in pbs . samples were then incubated overnight at 4c with specific rabbit polyclonal anti - human calreticulin antibodies ( santa cruz biotechnology , usa ) diluted in a pbs solution ( 1:250 ) containing 1% bsa , 0.05% triton , 1% nds , and 1% ovalbumin . the anti - calreticulin antibodies were directed against an antigen sequence corresponding to the lys - leu region of human calreticulin , presenting 81% homology with leech calreticulin . after 3 washes with pbs , samples were incubated 1 h at room temperature with anti - rabbit donkey antibody ( invitrogen , usa ) conjugated to alexa fluor 488 ( 1:2000 ) in a pbs solution containing 1% bsa , prior to mounting , the cell nuclei were counterstained by hoechst 33342 fluorescent dye ( 1 : 1000 , invitrogen usa ) for 10 min . the biotinylation of the recombinant human c1q ( prospecbio , usa ) was carried out by using the sulfo - nhs - ss - biotin kit ( pierce , usa ) according to the manufacturer s instructions . unreacted sulfo - nhs - ss - biotin was removed using the zeba desalt spin columns ( pierce , usa ) . biotinylated human c1q was immediately fixed onto a streptavidin column ( pierce , usa ) , previously equilibrated with 5 volumes of pbs 0.1 m . microglia protein extract ( 800 g ) was added in the column , incubated overnight at 4c , and rinsed 10 times with pbs 0.1 m . captured microglial cell proteins were eluted from the streptavidin - agarose with 5% 2-mercaptoethanol - pbs 0.1 m at 30c for 30 min . proteins were precipitated in 10% trichloroacetic acid / acetone at 20c for 45 min and centrifuged at 13000  g for 15 min . the protein pellet was washed in cold acetone , air dried and dissolved in lmmli buffer . two other columns were used for the negative controls : the first containing the biotinylated human c1q without any microglia protein extract , and the second containing only the microglia protein extract to evaluate the unspecific reaction between streptavidin and microglial cell components . the membrane was incubated for 30 min at rt in blocking solution ( 0.05% tween , milk powder 5% w / v in pbs ) and then overnight at 4c in rabbit polyclonal anti - human calreticulin antibodies ( santa cruz biotechnology , usa ) , diluted at 1:5000 in blocking solution containing 1% ovalbumin . after rinsing with pbs-0.05% tween , the membrane was incubated for 1 h at rt in secondary goat anti - rabbit polyclonal antibodies conjugated with horseradish peroxidase ( jackson immunoresearch , usa ) ( dilution 1:20000 ) . the signal was revealed using the ecl kit supersignal west dura chemoluminescent substrate ( pierce , usa ) on kodak x - omat ls film ( sigma - aldrich , usa ) . in vitro chemotaxis assays were performed by using the double - p assay as described by khidai et al . with minor modifications . thirty - five millimeter petri dishes were filled with 1 ml of a 0.5% agar and 1% gelatin solution . after drying , one well was filled with 50 l of purified microglial cells ( see above ) and the other with chemotactic factor or negative controls reagents . either vehicle ( non - transformed pischia pastoris culture supernatant ) or recombinant hmc1q ( rhmc1q ) produced in p. pastoris   alone or with soluble calr were used ( 8 l ) as chemotactic factors . for inhibitory chemotactic experiments , cells were pre - incubated for 1 h at rt either with rabbit polyclonal anti - human calreticulin antibodies ( 1:1000 ) or with rabbit igg isotype as negative control ( 1:1000 ) . the number of migrating cells was counted on a hemocytometer ( 3 different counts ) under an axioskop microscope ( zeiss , germany ) . in mammals , c1q exerts a chemotactic activity on dendritic cells through different receptors : a gc1qr recognizing the globular head of c1q and a cc1qr recognizing the collagen domain of c1q . the presence of a putative calreticulin ( cc1qr ) was investigated in the leech h. medicinalis . based on the h. medicinalis genome analysis , a sequence homologous to known calreticulin sequences was detected . the design of specific primers then allowed the amplification of a full - length mrna sequence for a calreticulin - related molecule , named hmcalr ( for hirudo medicinalis calreticulin ) . hmcalr mrna ( genbank kf709537 ) encodes a 420-amino - acid sequence with a theoretical molecular weight of 48 610 da . the protein sequence presents a putative signal peptide and a calreticulin domain in met - ser and in val - ile amino - acid regions , respectively ( figure 1a ) . in addition , the analysis of hmcalr protein using a blast - p program shows a high conservation with human calreticulin , particularly in different domains that are already described for mammalian calreticulins . as presented in figure 1b , hmcalr might possess a single disulfide bridge ( cys - cys ) and contains a single histidine residue ( his ) . these residues were also described in an n - domain of the human mature calreticulin in cys - cys and his positions , respectively , ( numerated from the signal peptide - free sequence ) and were demonstrated as essential for the chaperoning function . the hmcalr sequence is also proline - rich and exhibits 2 repeated amino - acid sequences  repeats a ( pxxixdpdaxkpedwde ) and b ( gxwxppxixnpxyx )  as observed in the human form s p - domain ( figure 1b ) . in leeches , some non - essential amino acids have been substituted with different ones having equivalent physicochemical properties ( glu instead of asp in human repeat a ; and val instead of ile in human repeat b ) . finally , the high conservation of numerous acidic residues in hmcalr suggests the presence of a c - domain , as in mammals . thus hmcalr presents sequence similarities with mammalian calreticulins and also with other forms from vertebrate , protostomian , and diploblastic species , as revealed by a neighbor - joining phylogenetic tree using selected sequences among them ( figure 2 ) . depending on the 200 highest similarities between calreticulin molecules and hmcalr , some were chosen to check the evolutive position of hmcalr according to species . the distribution of sequences in the cladogram respects the species organization between vertebrates and protostomians . the neighbor - joining phylogenetic tree shows that leech calreticulin is positioned in lophotrochozoan organisms sharing the same origin with eisenia andrei , another annelid organism , and also close to mollusks . arthropods have calreticulins that form a distinct group in protostomians , which is relevant in their distinction from the lophotrochozoans . otherwise , although diploblastic organisms , cnidarians present higher similarities in calreticulin sequences with lophotrochozoans than with those of arthropods . finally , vertebrate calreticulins are positioned in a distinct clade . cells expressing hmcalr transcripts in the leech nervous system were investigated by specific fluorescence in situ hybridization ( fish ) on injured nerve cords after different times . the transcripts were detected in only some scattered microglial cells among all microglial cells ( blue nuclei ) in the connectives 24 h after lesion . no specific signal was detected with sense riboprobes used as negative control ( figure 3a ) . cells containing calreticulin protein were investigated by immunohistochemistry with rabbit polyclonal anti - human calreticulin antibodies on whole mounted injured nerve cords immediately after dissection ( figure 3b3b4 ) and 24 h after lesion ( figure 3c , 3d ) . the microglia nuclei were simultaneously counterstained by using a hoechst dye ( blue nuclei ) . immediately following the crush ( t=0 ) , calreticulin staining ( green ) was detected in only some microglial cells in the connectives among the whole microglia ( blue nuclei ) . the hmcalr - positive microglia appeared spindle - shaped and very elongated , suggesting cell mobility . only a few microglial cells in the connective seemed to be positive for calreticulin , but the analyses of successive focal plans in the connectives allowed the observation of more microglial cells among the whole connective ( figure 3b3b4 ) . twenty - four hours after injury , when the acute microglia migration declines , the calreticulin staining was detected in some ramified microglial cells distributed in the injured connectives ( figure 3c ) . in addition , the labeling was mainly localized around the nucleus and seemed to be vesicular in the microglia cytoplasm ( figure 3c ) . negative controls performed using the secondary antibodies alone did not show any signal ( figure 3d ) . calreticulin ( alias cc1qr ) and c1qbp ( alias gc1qr ) bind to the collagen domain and globular head , respectively , of c1q . because previous results demonstrated an interaction between hmc1q and hmc1qbp in the leech cns , co - purification experiments once biotinylated , human c1q was immobilized on a streptavidin column and incubated with leech microglia protein extracts ( figure 4a ) . following the elution , the interactants of c1q were analyzed by western blotting using polyclonal anti - human calr antibodies ( figure 4b ) that specifically recognized a unique ~55 kda molecule ( figure 4b , lane1 ) , probably corresponding to hmcalr due to the 81% identity of both leech and human antigenic sequences . this analysis presents a shift between the observed size and the expected one ( 48 610 da for hmcalr containing the signal peptide ) . this difference of migration in sds - page was already reported for other calreticulins [ 3639 ] and is probably due to the high content of acidic residues in the c - terminal end of the sequence . in the first negative control , when microglia protein extracts were incubated on a streptavidin column alone ( figure 4b , lane 2 ) , no signal was obtained , showing that anti - human calr antibodies do not react with leech microglial proteins non - specifically captured by streptavidin column . in the second negative control , when the biotinylated human c1q was loaded alone on streptavidin column ( figure 4b , lane 3 ) , no immunoreactivity was detected , indicating that the anti - human calr antibodies do not react with the human c1q . no signal was observed in negative controls using only the secondary antibody ( figure 4c ) . these results gave evidence of a specific interaction between human c1q and hmcalr protein present in leech microglia protein extract . c1q has been shown to recruit leech microglial cells in a dose - dependent manner . in the present study , leech microglial cells in l-15 medium were demonstrated to migrate towards the recombinant form of hmc1q ( rhmc1q ) about 6-fold more than towards vehicle ( figure 5 ) . the pre - incubation of microglial cells with rabbit polyclonal anti - human calreticulin antibodies showed a significant decrease of the rhmc1q chemotactic effect ( from 6-fold to 2.5-fold ) , whereas no significant inhibitory effect was detected with the rabbit igg isotype as negative control . the microglial recruitment was also strongly reduced ( from 6-fold to 2.3-fold ) when rhmc1q was used in association with soluble calr ( scalr ) on cells in l-15 medium . in mammals , c1q exerts a chemotactic activity on dendritic cells through different receptors : a gc1qr recognizing the globular head of c1q and a cc1qr recognizing the collagen domain of c1q . the presence of a putative calreticulin ( cc1qr ) was investigated in the leech h. medicinalis . based on the h. medicinalis genome analysis , a sequence homologous to known calreticulin sequences was detected . the design of specific primers then allowed the amplification of a full - length mrna sequence for a calreticulin - related molecule , named hmcalr ( for hirudo medicinalis calreticulin ) . hmcalr mrna ( genbank kf709537 ) encodes a 420-amino - acid sequence with a theoretical molecular weight of 48 610 da . the protein sequence presents a putative signal peptide and a calreticulin domain in met - ser and in val - ile amino - acid regions , respectively ( figure 1a ) . in addition , the analysis of hmcalr protein using a blast - p program shows a high conservation with human calreticulin , particularly in different domains that are already described for mammalian calreticulins . as presented in figure 1b , hmcalr might possess a single disulfide bridge ( cys - cys ) and contains a single histidine residue ( his ) . these residues were also described in an n - domain of the human mature calreticulin in cys - cys and his positions , respectively , ( numerated from the signal peptide - free sequence ) and were demonstrated as essential for the chaperoning function . the hmcalr sequence is also proline - rich and exhibits 2 repeated amino - acid sequences  repeats a ( pxxixdpdaxkpedwde ) and b ( gxwxppxixnpxyx )  as observed in the human form s p - domain ( figure 1b ) . in leeches , some non - essential amino acids have been substituted with different ones having equivalent physicochemical properties ( glu instead of asp in human repeat a ; and val instead of ile in human repeat b ) . finally , the high conservation of numerous acidic residues in hmcalr suggests the presence of a c - domain , as in mammals . thus hmcalr presents sequence similarities with mammalian calreticulins and also with other forms from vertebrate , protostomian , and diploblastic species , as revealed by a neighbor - joining phylogenetic tree using selected sequences among them ( figure 2 ) . depending on the 200 highest similarities between calreticulin molecules and hmcalr , some were chosen to check the evolutive position of hmcalr according to species . the distribution of sequences in the cladogram respects the species organization between vertebrates and protostomians . the neighbor - joining phylogenetic tree shows that leech calreticulin is positioned in lophotrochozoan organisms sharing the same origin with eisenia andrei , another annelid organism , and also close to mollusks . arthropods have calreticulins that form a distinct group in protostomians , which is relevant in their distinction from the lophotrochozoans . otherwise , although diploblastic organisms , cnidarians present higher similarities in calreticulin sequences with lophotrochozoans than with those of arthropods . finally , vertebrate calreticulins are positioned in a distinct clade . cells expressing hmcalr transcripts in the leech nervous system were investigated by specific fluorescence in situ hybridization ( fish ) on injured nerve cords after different times . the transcripts were detected in only some scattered microglial cells among all microglial cells ( blue nuclei ) in the connectives 24 h after lesion . no specific signal was detected with sense riboprobes used as negative control ( figure 3a ) . cells containing calreticulin protein were investigated by immunohistochemistry with rabbit polyclonal anti - human calreticulin antibodies on whole mounted injured nerve cords immediately after dissection ( figure 3b3b4 ) and 24 h after lesion ( figure 3c , 3d ) . the microglia nuclei were simultaneously counterstained by using a hoechst dye ( blue nuclei ) . immediately following the crush ( t=0 ) , calreticulin staining ( green ) was detected in only some microglial cells in the connectives among the whole microglia ( blue nuclei ) . the hmcalr - positive microglia appeared spindle - shaped and very elongated , suggesting cell mobility . only a few microglial cells in the connective seemed to be positive for calreticulin , but the analyses of successive focal plans in the connectives allowed the observation of more microglial cells among the whole connective ( figure 3b3b4 ) . twenty - four hours after injury , when the acute microglia migration declines , the calreticulin staining was detected in some ramified microglial cells distributed in the injured connectives ( figure 3c ) . in addition , the labeling was mainly localized around the nucleus and seemed to be vesicular in the microglia cytoplasm ( figure 3c ) . negative controls performed using the secondary antibodies alone did not show any signal ( figure 3d ) . calreticulin ( alias cc1qr ) and c1qbp ( alias gc1qr ) bind to the collagen domain and globular head , respectively , of c1q . because previous results demonstrated an interaction between hmc1q and hmc1qbp in the leech cns , co - purification experiments once biotinylated , human c1q was immobilized on a streptavidin column and incubated with leech microglia protein extracts ( figure 4a ) . following the elution , the interactants of c1q were analyzed by western blotting using polyclonal anti - human calr antibodies ( figure 4b ) that specifically recognized a unique ~55 kda molecule ( figure 4b , lane1 ) , probably corresponding to hmcalr due to the 81% identity of both leech and human antigenic sequences . this analysis presents a shift between the observed size and the expected one ( 48 610 da for hmcalr containing the signal peptide ) . this difference of migration in sds - page was already reported for other calreticulins [ 3639 ] and is probably due to the high content of acidic residues in the c - terminal end of the sequence . in the first negative control , when microglia protein extracts were incubated on a streptavidin column alone ( figure 4b , lane 2 ) , no signal was obtained , showing that anti - human calr antibodies do not react with leech microglial proteins non - specifically captured by streptavidin column . in the second negative control , when the biotinylated human c1q was loaded alone on streptavidin column ( figure 4b , lane 3 ) , no immunoreactivity was detected , indicating that the anti - human calr antibodies do not react with the human c1q . no signal was observed in negative controls using only the secondary antibody ( figure 4c ) . these results gave evidence of a specific interaction between human c1q and hmcalr protein present in leech microglia protein extract . c1q has been shown to recruit leech microglial cells in a dose - dependent manner . in the present study , leech microglial cells in l-15 medium were demonstrated to migrate towards the recombinant form of hmc1q ( rhmc1q ) about 6-fold more than towards vehicle ( figure 5 ) . the pre - incubation of microglial cells with rabbit polyclonal anti - human calreticulin antibodies showed a significant decrease of the rhmc1q chemotactic effect ( from 6-fold to 2.5-fold ) , whereas no significant inhibitory effect was detected with the rabbit igg isotype as negative control . the microglial recruitment was also strongly reduced ( from 6-fold to 2.3-fold ) when rhmc1q was used in association with soluble calr ( scalr ) on cells in l-15 medium . as we previously described in the medicinal leech , the c1q molecule called hmc1q is important for the recruitment of microglial cells within the hours following a lesion of the nerve cord . we also reported that hmc1q exerts its chemotactic functions on leech microglia by recognizing at least one receptor homologous to human gc1qr ( c1qbp ) . in mammals , the interaction between c1q and receptors such as gc1qr ( c1qbp ) and calreticulin ( cc1qr ) has so far been reported for dendritic cell chemoattraction   but never for microglia recruitment . we investigated in the leech cns the existence of a calreticulin molecule and its involvement in microglial accumulation at the site of injury . in the present report , the characterization of hmcalr in the medicinal leech showed high sequence similarities with other calreticulins as presented in the neighbor - joining phylogenetic tree of selected calreticulins . that corroborates the fact that calreticulin ( calr or crt ) is widely distributed in metazoa . the result respects the actual phylogenetic systematics , except for the cnidarian sequences that appeared close to lophotrochozoan ones . then we focused on a comparison of the different domains between the leech and the human forms . human calreticulin is a 46-kda chaperone protein containing 3 structurally and functionally distinct domains ( n- , p- , and c - domains ) [ 4042 ] . the n- and p - domains in mammalian calreticulins are associated to the lectin - like chaperoning function , whereas the c - domain is involved in ca storage in the endoplasmic reticulum through its highly acidic properties . the alignment between leech and human sequences shows that they share several of these structural features . the leech molecule hmcalr presents all the critical residues determining the 3 domains , including cysteines , probably forming a disulfide bridge , and the essential histidine responsible for the chaperoning function . despite the lack of a structural analysis of hmcalr in this report , the high conservation of critical residues suggests some similarities with human calreticulin for domain - dependent functions . among these functions , calreticulin plays an important role in immune responses by acting as a receptor for c1q , mannose - binding lectins , and ficolins . the presence of hmcalr mrna and hmcalr protein in only some microglial cells along the nerve cord led us to better understand the involvement of this molecule in neuro - immune processes . beside the use of calreticulin in c1q - dependent chemoattraction of dendritic cells , immune functions for calreticulin have ever been reported in the literature . during a viral infection in crustaceans , for example , c1q receptors such as calreticulin or gc1qr are differentially produced in crustacean immune cells and help regulate the replication of viruses . in mammals , the presence of calreticulin at the surface of cells can induce the attraction of macrophages and dendritic cells through a c1q - dependent interaction . thus , the exposure of calreticulin can be used as a strong phagocytic signal leading to the recognition of calreticulin cells . in this context , parasites such as trypanosoma cruzi expose calreticulin to facilitate its recognition by host macrophages and optimize its internalization . recently , they were also suggested on nerve cells where calreticulin exposure on neurons was required to allow their in vitro phagocytosis by bv2 microglia cell line . in the leech cns , even if hmcalr has not been detected in neurons so far , the affinity purification experiments from microglial protein extracts demonstrated the interaction of c1q with hmcalr through its specific detection using the polyclonal anti - human calreticulin antibodies . then , both results in chemotaxis assays clearly indicated that this hmc1q / hmcalr interaction contributes to c1q - dependent chemotactic properties on microglia . in this report , due to hmcalr staining in a few microglial cells observed at lesions , the use of such a molecule as a receptor for hmc1q thus , independently of the maturity of microglial cells , the limited exposure of hmcalr might be relevant to other known receptors . indeed , we previously showed that the presence of leech gc1qr ( hmc1qbp ) concerned only a subpopulation of microglial cells accumulated at lesions . our data have never permitted the observation of a dual localization of both hmc1qbp and hmcalr in the same microglial cells . therefore , the action of hmc1q allows the chemoattraction of distinct microglia subtypes at the lesion site . further studies will aim to specify their respective functions towards the damaged neurons . in mammals , c1q is the first component of the classical complement pathway . in early brain development , it mediates the cns synapse elimination during neurogenesis . in adult cns disorders , c1q is also released by activated microglia to maintain and regulate their activation . among the mediators expressed by microglial cells and neurons , c1q seems to be a key molecule in neuroinflammatory diseases   and in various neurodegenerative pathologies such as alzheimer disease [ 5862 ] . importantly , some studies recently showed that c1q also has complement cascade - independent activities . c1q exerts ( independently of its interaction with c1r and c1s ) a neuroprotective effect against amyloid- plaques . of interest , our data in the leech suggest the importance of c1q in a non - classical complement pathway because the analysis of h. medicinalis genome revealed neither c1r- nor c1s - related molecule . by taking into account the importance of microglia accumulation at a lesion to engage the axonal sprouting in the leech cns in addition , other chemotactic factors allow the microglial accumulation at a lesion in the leech cns   and also use specific receptors . thus , hmc1q , as well as hmil-16 and hmemapii , allow the accumulation of microglia . so we must now consider the leech microglia as a mixture of reactive microglia subpopulations where distinct sets can be recruited through the influence of different chemotactic molecules . in this context , microglia probably contribute to different pro- and/or anti - inflammatory mechanisms by communicating with other glial cells and neurons . further studies will investigate the discrimination of microglia subpopulations that are recruited to the lesions . they will specify the time course of the accumulation and the specific functions of subpopulations . the complexity of immune responses following a cns damage is enhanced by multiple cell origin and activation states of microglia . in mammals , the resident microglia that result from the invasion processes during embryonic neurogenesis are helped by infiltrated bone marrow - derived cells and circulating monocytes during cns diseases . nevertheless , despite in vitro cell analyses , morphological and/or histological in vivo studies besides the cell origin , different functional profiles can simultaneously act and exhibit pro - inflammatory features ( classical activation , m1 ) or anti - inflammatory features ( alternative activation , m2 ) depending on the recruitment of specific microglia subtypes at lesions . in this context the medicinal leech as a model in nerve repair has been used for decades because it represents a new insight into study of the cell processes engaged during the axonal sprouting . in addition , the leech microglia has been demonstrated to be essential for efficient nerve repair . because they are not supported by a significant infiltration of blood cells , microglial cells in the leech represent an alternate model of interest in comparative microglial studies . according to dynamic and functional properties , the impact of microglia recruitment under the influence of hmcalr / hmc1q interaction will be studied to specify the microglial contribution in cns inflammatory regulation .
backgroundthe medicinal leech is considered as a complementary and appropriate model to study immune functions in the central nervous system ( cns ) . in a context in which an injured leech s cns can naturally restore normal synaptic connections , the accumulation of microglia ( immune cells of the cns that are exclusively resident in leeches ) has been shown to be essential at the lesion to engage the axonal sprouting . 
 hmc1q ( hm for hirudo medicinalis ) possesses chemotactic properties that are important in the microglial cell recruitment by recognizing at least a c1q binding protein ( hmc1qbp alias gc1qr).material / methodsrecombinant forms of c1q were used in affinity purification and in vitro chemotaxis assays . 
 anti - calreticulin antibodies were used to neutralize c1q - mediated chemotaxis and locate the production of calreticulin in leech cns.resultsa newly characterized leech calreticulin ( hmcalr ) has been shown to interact with c1q and participate to the hmc1q - dependent microglia accumulation . 
 hmcalr , which has been detected in only some microglial cells , is consequently a second binding protein for hmc1q , allowing the chemoattraction of resident microglia in the nerve repair process.conclusionsthese data give new insight into calreticulin / c1q interaction in an immune function of neuroprotection , suggesting another molecular target to use in investigation of microglia reactivity in a model of cns injury .