mast cells are integrally involved in cellular based immune responses to pathogens as well as inflammatory reactions prompted by pathogens or toxins [ 1 , 2 ] and have been suggested for several years to play a part in the acute phase of multiple sclerosis ( ms ) [ 38 ] .
albeit the mechanisms by which mast cells influence ms are yet to be fully understood , trypsin - like proteases released from degranulating mast cells have been shown to trigger demyelination in a mouse model for ms - experimental autoimmune encephalomyelitis ( eae ) .
( 2000 ) , furthermore , support that mast cells are involved in the pathogenesis of eae .
the same group has more recently suggested a significant role for mast cells in activation of inflammasomes localized within meninges .
in contrast , other groups have challenged the contribution of mast cells in eae [ 1113 ] . in particular , feyerabend and colleagues
albeit targeting mast cells as a viable approach to alleviate the disease remains debatable , the contribution of mast cell - derived proteases in eae is still largely uninvestigated .
one of the many mast cell - expressed proteases that potentially may account for the contribution of mast cells to ms / eae is mouse mast cell protease 4 ( mmcp-4 ) , a -chymase predicted to be the murine functional counterpart to the single human chymase ( cma1 ) based on deduced amino acid sequence , tissue localization , and serglycin storage dependence [ 14 , 15 ] . in support for a role of this chymase in mast cell - dependent inflammatory conditions , mmcp-4 plays a protective role in a mouse model of mechanically induced cerebral trauma , yet it is detrimental in lung inflammation and immune complex - induced glomerulonephritis [ 17 , 18 ] . in further support for a role of mmcp-4 in regulating inflammatory mediators , our group has reported that mmcp-4 generates endothelin-1 ( et-1 ) from its precursor big - et-1 [ 19 , 20 ] and that mmcp-4 knockout ( ko ) mice display a 40% reduction in pulmonary et-1 levels when compared to wild type ( wt ) congeners .
the role of et-1 as a marker in the etiology of ms has only been explored in a limited fashion .
( 2001 ) reported a significant increase in et-1 plasma levels in untreated ms patients , an observation confirmed by pache and colleagues ( 2003 ) . in treated ms patients
however , in further support for a role of et-1 in ms , et-1 was shown to be overexpressed in a murine model of eae .
based on the above - suggested links between et-1 and ms and between chymase and et-1 generation , respectively , we here asked whether chymase might have a role in eae and whether that enzyme in this experimental setting has a regulatory effect on et-1 production .
indeed , the findings presented here suggest that mmcp-4 has a significant detrimental impact on the course of eae and plays role in the generation of et-1 in this mouse model for ms .
the impact of mmcp-4 in eae introduces a potential role for mast cell chymase in ms and thereby identifies the inhibitors of this particular enzyme as potential targets for therapy of ms .
c57bl/6 mice ( i.e. , wild type ( wt ) ) were purchased from charles river canada ( montral , qc , canada ) and housed in our local facility .
the mmcp-4 ko mice have been backcrossed for over 10 generations with c57bl/6 congeners and are therefore highly congenial with the later strain .
finally , the genotype of mmcp-4 ko mice used in the present study was confirmed by polymerase chain reaction ( pcr ) ( as shown in supplementary figure  1 in supplementary material available online at http://dx.doi.org/10.1155/2016/9797021 ) via the use of primers described in supplemental table  1 .
we had previously reported , in mmcp-4 ko mice in vivo as well as in tissues or mastocytes derived from this mouse strain , the complete loss of chymase - dependent hydrolytic activity [ 19 , 20 ] .
all animals were kept at constant room temperature ( 23c ) and humidity ( 78% ) under a controlled 1014  h light / dark cycle .
animal care and experimentation were approved by ethics committee on animal research of the universit de sherbrooke in accordance with the guidelines of the canadian council on animal care .
induction of eae was performed according to the protocol of miller and karpus ( 2007 ) . in brief , a 1  :  1 emulsion mixture of myelin oligodendrocyte glycoprotein ( mog3555 ) ( genemed synthesis inc .
, san antonio , tx , usa ) and complete freund 's adjuvant ( cfa ) ( sigma - aldrich , st .
louis , mo , usa ) supplemented with 100  g of heat - killed mycobacterium tuberculosis h37ra ( difco laboratories , detroit , mi , usa ) was prepared .
female mice , at 810 weeks old , were subcutaneously injected in two sites ( 100  l by site ) adjacent to the tail with the emulsion .
campbell , ca , usa ) was administered intraperitoneally on the same day of immunization .
mice were scored daily with the following scale to assess clinical scores : 0 , no sign of clinical disease ; 0.5 , partial tail paralysis ( loss of tip tail tonus ) ; 1 , tail flaccidity or hind limb weakness ; 2 , limp tail and weakness in limb ; 3 , partial hind limb paralysis ; 4 , total hind limb paralysis ; and 5 , moribund state or death .
the immunized mice were anesthetized by intraperitoneal injection of 2,2,2- tribromoethanol ( avertin ) ( approximately 240  mg / kg ) ( sigma - aldrich , st .
louis , mo , usa ) , prepared in tert - amyl alcohol and diluted in 0,9% saline solution .
the mice were then perfused with ice - cold pbs buffer ( wisent , st .
bruno , qc , canada ) , and the spleen , cervical , and thoracic spinal cords and left and right brain hemispheres were collected and stored at 80c immediately , whereas lumbar spinal cords were placed in 10% buffered formalin phosphate ( fisher scientific , waltham , ma , usa ) before being embedded in paraffin and cut into 5  m sections .
slides were deparaffinised in xylene ( electron microscopy sciences , hatfield , pa , usa ) and hydrated in 100 , 95 , and 70% ethanol gradient followed by water .
slides were incubated 2 - 3 minutes at room temperature in toluidine blue ( sigma - aldrich , st .
louis , mo , usa ) working solution prepared by dilution of stock solution ( 1% in ethanol 70% ) in sodium chloride 1% ph 22.5 .
the slides were rinsed in water and quickly dehydrated in 95100% ethanol before being rinsed with xylene and coverslipped with permount ( fisher scientific , ottawa , on , canada ) .
slides were incubated overnight at 56c in 0.1% luxol fast blue solution ( electron microscopy sciences , hatfield , pa , usa ) in 95% alcohol w / acetic acid .
the slides were then rinsed in distilled water before being differentiated in lithium carbonate 0.05% and in 70% ethanol for approximately 30 seconds each , since the grey matter is clear and white matter is sharply defined .
the slides were rinsed in water and dehydrated in 100% ethanol before being cleared in xylene and coverslipped with permount .
all histological slides were scanned with a nanozoomer 2.0-rs digital slide scanner ( hamamatsu photonics , shizuoka , japan ) before being treated with the ndp.view2 viewing software and paint.net software and staining density was quantified by imagej 1,49v ( wayne rasband , nih , usa ) .
prior to measuring immunofluorescence , with a sequenze slide rack and coverplate system ( ted pell inc , redding , ca , usa ) , an antigen unmasking was performed by a 10 minutes ' incubation in 10  mm sodium citrate buffer ph 6.0 ( sigma - aldrich , st .
the slides were then washed in 0.1% triton x-100 in pbs solution and blocked in 5% fetal bovine serum ( fbs ) supplemented with 0.1% triton x-100 in pbs for one hour before being incubated overnight at 4c with primary antibody ( 1  :  1000 ) , against the rabbit anti - glial fibrillary acidic protein ( gfap ) ( cedarlane , burlington , on , canada ) or against the rabbit anti - ionized calcium binding adaptor molecule 1 ( iba1 ) ( wako , osaka , japan ) . a 2 hours ' incubation at room temperature with the secondary antibody ( 1  :  2000 ) , alexa fluor 488 affinipure goat anti - rabbit igg ( h + l ) ( jackson immunoresearch laboratories inc .
the slides were mounted with dapi fluoromount - g ( southernbiotech , birmingham , al , usa ) and photomicrograph pictures were taken with retiga srv mono cooled numerical camera attached to zeiss axioskop 2 microscope .
the pictures were stitched with adobe photoshop cs3 , and stain density was quantified with image - pro plus 5.1 ( media cybernetics inc . ,
the left parts of brain from healthy or 1 , 2 , or 3 weeks post - eae - induced mice were homogenized in a chloroform  :  methanol ( 1  :  4 ) solution and then purified on a dsc-18 solid phase extraction column ( supelco , bellefonte , pa , usa ) and eluted in acetonitrile  :  water  :  trifluoroacetic acid ( acn 60%  :  h2o 40%  :  tfa 0,1% ) .
the collected eluates were then speed vac - dried overnight before reconstitution in pbs supplemented with 1/32 mouse plasma and endogenous et-1 was measured by quantikine elisa kit from r&d systems ( r&d systems , minneapolis , mn , usa ) according to the manufacturer 's instructions .
briefly , frozen thoracic spinal cords were weighed and homogenized in 0.5  ml of ice - cold lysis buffer ( cell signaling technology , beverly , ma , usa ) supplemented with protease inhibitors ( roche diagnosis , mannheim , germany ) by rapid agitation for 2 minutes in the presence of 3  mm stainless beads .
the tissue lysate was centrifuged for 20 minutes at 13,000  g at 4c , and the supernatant was transferred to a new tube .
the tissue levels of ifn were determined using murine elisa development kits ( peprotech , rocky hill , nj , usa ) , according to the manufacturer 's instructions . the level of ifn was reported as pg / mg of tissue .
rna from the right brain hemisphere derived from healthy or 1 or 2 weeks post - eae - induced mice were extracted using ribozol reagent ( amresco inc . ,
baker , central valley , pa , usa ) was added to each tube per 1  ml of ribozol and incubated at room temperature for 3 minutes followed by centrifugation at 12,000  g for 15 minutes at 4c .
nonopaque supernatants were collected and 500  l of isopropanol ( fisher scientific , ottawa , on , canada ) was added for rna precipitation and incubated 10 minutes at room temperature followed by centrifugation at 12,000  g for 10 minutes at 4c .
pellets were washed with addition of 1  ml ethanol 75% followed by 7,500  g centrifugation at 4c for 5 minutes before being redissolved in 50  l of depc water and an incubation at 55c for 10 minutes .
cdna was synthesized using oligo(dt)1218 primers ( invitrogen , carlsbad , ca , usa ) , dntps mix 10  m each ( thermo scientific , waltham , ma , usa ) , in superscript iii buffer with dtt , rnaseout , and superscript iii ( invitrogen , carlsbad , ca , usa ) .
quantitative pcr was performed for actin and mmcp-4 by monitoring in real time the fluorescence increase of the sybr green in the perfecta sybr green supermix , low rox ( quanta biosciences , gaithersburg , md , usa ) using the mx3000p multiplex quantitative pcr system ( agilent technologies , santa clara , ca , usa ) .
primers ( idt , coralville , ia , usa ) were used at final concentration of 50  nm per primer and sequences were designed as follows :  mmcp-4 f : 5-ctctctccaagctgtgaccgac-3 ,  mmcp-4 r : 5-ctatgagctccaagggtgaca-3 ,  -actin f : 5-gatcaagatcattgctcctcctgagc-3 ,  -actin r : 5-gcagctcagtaacagtccgcctag-3.mmcp-4 ko mice samples were tested as negative controls .
as -actin levels were stable between healthy and immunized mice , the latter mrna was used as internal control for normalization and relative expression of chymase mmcp-4 was calculated using the 2 method . mmcp-4
f : 5-ctctctccaagctgtgaccgac-3 , mmcp-4 r : 5-ctatgagctccaagggtgaca-3 , -actin f : 5-gatcaagatcattgctcctcctgagc-3 , -actin r : 5-gcagctcagtaacagtccgcctag-3.
all statistical analyses were conducted using graphpad prism 6 software ( graphpad software , la jolla , ca , usa ) .
value was below 0.05 and determined using one - way anova kruskal - wallis followed by bonferroni for eae clinical score or based on one - way or two - way anova and multiple student 's t - test for all other analyses .
an emulsified mixture of mog3555 in cfa was subcutaneously injected in wt or mmcp-4 ko mice .
wt mice developed the disease approximately after 9 days whereas the mmcp-4 ko mice demonstrated a substantial delay in the appearance of clinical symptoms , being detectable starting from ~day 12 after injection ( figure 1(a ) ) .
moreover , the development of severe disease ( disease score 2.5 ) was markedly delayed in mmcp-4 ko mice up until the third week posttreatment as indicative of tail and back limb weaknesses .
however , 4 weeks after eae , wt and mmcp-4 ko mice exhibited similar disease scores with no evidence of total hind limb paralysis .
a summation of the total clinical scores , on the other hand , supported a significant role of mmcp-4 as a pathogenic factor in eae ( figure 1(b ) ) , even up to the 4th week of observations ( wt 41.64  3.02 ; mmcp-4 ko 30.93  2.48 , p < 0.05 , n = 7 mice ) . to assess , on the other hand , whether mmcp-4 has an impact on the immune response , spleen weights of wt and mmcp-4 ko mice were monitored up to 4 weeks after immunization
however , as seen in figure 2 , spleen weights did not differ between the two genotypes except in healthy mice where it was significantly increased in mmcp-4 ko congeners ( p < 0.05 ) . in response to spinal cord insults ,
gfap ( an intermediate filament protein expressed by astrocytes and ependymal cells among others ) is upregulated in the cns . as another sign of cns damage
, the reactive microglial response can be measured by the extent of upregulation of iba1 . to evaluate whether mmcp-4 can influence the levels of these markers of cns damage , the spinal cords of 2 weeks after eae immunized mice were extracted and stained for gfap and iba1 ( figure 3(a ) ) .
mmcp-4 ko mice and wt littermates showed no significant differences in percent level of astrogliosis ( gfap staining intensity ) and microgliosis ( iba1 staining intensity ) in the total area of the lumbar spinal cord or in the white matter ( figure 3(b ) ) .
in contrast , significant reductions in percentages of microgliosis and astrogliosis were found in the grey matter of mmcp-4 ko mice when compared to wt congeners , 2 weeks after eae immunization ( in wt and mmcp-4 ko mice , respectively . ; astrogliosis in percentage : 0.87  0.17 and 0.30  0.04 , p < 0.05 ; microgliosis in percentage : 1.61  0.18 and 0.55  0.12 , p < 0.01 ) ( figure 3(b ) ) . to assess whether the absence of mmcp-4 has an influence on the number of mast cells , lumbar spinal cords from wt and mmcp-4 ko mice were stained with toluidine blue , both at baseline and after induction of eae .
as seen in figure 4 , the numbers of mast cells were similar in spinal cords from wt and mmcp-4 ko mice at the baseline state ( healthy ) and 2 - 3 weeks after induction of eae .
notably though , there was a significant elevation of the numbers of mast cells 1 week after eae ( in wt and mmcp-4 ko mice , respectively ; in percentage , healthy state : 2.30  0.07 and 2.47  0.17 , 1 week after eae : 2.93  0.14 and 3.74  0.27 , p < 0.05 , 2 weeks after eae : 2.73  0.25 and 2.93  0.20 , and 3 weeks after eae : 3.20  0.13 and 3.29  0.17 ) . to assess whether chymase has an impact on the levels of intact myelin , lumbar spinal cords from wt and mmcp-4 ko mice were extracted and stained with luxol fast blue , a dye that stains myelin ( figure 5 ) .
quantification of the luxol fast blue staining intensity revealed a significantly higher myelin content in mmcp-4 ko mice as compared with wt congeners , in both nonimmunized ( healthy ) mice and immunized mice 1 week after eae induction ( figure 6 ) ( in percentage , healthy wt mice : 31.62  0.46 and healthy mmcp-4 ko mice : 39.70  1.03 , p < 0.001 ; wt 1 week after eae : 27.37  0.29 and mmcp-4 ko mice 1 week after eae : 33.88  0.50 , p < 0.001 ) .
a subsequent decline in spinal cord myelin was seen in both wt and mmcp-4 ko mice 2 to 3 weeks after immunization as well as a loss of significant differences in myelin content between the two strains of mice ( figure 6 ) . to investigate if eae is associated with an induction of the mmcp-4 gene , right brain homogenates derived from wt mice were analysed for mmcp-4 mrna levels by rt - qpcr . as seen in figure 7(a ) , a 2.2-fold increase in mmcp-4 mrna expression ( as compared with baseline levels in healthy mice ) was found in wt mice one week after eae immunization but not at later time points .
finally , eae immunization promoted a twofold increase in mature et-1 levels in left brain homogenates of wt but not mmcp-4 ko mice , one but not two weeks after eae when compared to healthy wt mice ( figure 7(b ) ) . to investigate the immune response to eae induction
, we proceeded to elisa quantification of ifn in thoracic spinal cord in healthy and 1 or 2 weeks after eae .
as shown in figure 7(c ) , the levels of this cytokine are increased after eae compared to healthy basal levels in both strains of mice , but significantly only in wt mice 2 weeks after eae ( quantities in pg / mg of tissue , healthy wt mice : 20.77  1.84 ; wt 1 week after eae : 23.30  2.11 ; wt 2 weeks after eae : 32.07  2.11 , p < 0.001 and healthy mmcp-4 ko mice : 19.23  1.65 ; mmcp-4 ko mice 1 week after eae : 24.66  4.24 ; mmcp-4 ko mice 2 weeks after eae : 26.47  3.39 ) .
the main results of the present study are that mmcp-4 ko mice subjected to eae show a reduced clinical score as well as brain levels of immunoreactive et-1 when compared to wt congeners . in addition , the ko mice show reduction in percentages of microgliosis and astrogliosis as well as grey matter alterations . these results therefore support our hypothesis that this particular chymase isoform plays a significant role in the early development of ms in the mouse eae model . in our hands
, the ko of mmcp-4 significantly reduced clinical scores up until 21 days after eae induction .
it is noteworthy that mature mast cells can express numerous tryptases ( having trypsin - like cleavage specificity ) and chymases ( having chymotrypsin - like cleave specificities ) as well as other types of proteases . out of these , mmcp-4 was shown here to have a major impact on the course of eae development .
this suggests that mmcp-4 accounts to a major extent for the demyelination attributed to mast cells in eae .
this does not exclude , on the other hand , the involvement of other mast cell - derived mediators in this mouse model of ms .
it is also of interest that eae prompted a significant increase in et-1 brain levels one week after eae induction in wt but not in mmcp-4 ko mice .
notably , brain levels of this potent vasoactive peptide progressively returned to basal levels on the second and third weeks after eae ( results not shown for the 3rd week ) .
these results suggest that et-1 may be either an early marker or , alternatively , an early mediator of the inflammatory reaction occurring in the eae model .
( 2001 ) reported that an eta antagonist , bq-123 , reduced the duration of paralysis , in eae - induced rats .
the same authors also reported high levels of immunoreactive et-1 in spinal cord - localized inflammatory and neuroglial cells .
( 2014 ) have recently shown that et-1 is a negative regulator of oligodendrocyte progenitor cell - dependent repair of demyelinated lesions . in support of the latter observation ,
myelin contents were , in the present study , found to be higher in naive and even one week after eae immunized mmcp-4 ko mice than in their wt congeners .
thus , interfering with the chymase - dependent production of et-1 in ms may be a relevant therapeutic strategy in autoimmune diseases such as ms .
noteworthy , chymase inhibitors have been shown to possess anti - inflammatory and antiproliferative properties in other settings [ 3437 ] .
albeit still controversial , one should also note the beneficial effects of et-1 antagonists on cerebral blood flow of ms patients .
thus , et-1 antagonists may have beneficiary effects not only by repressing cellular events in eae but by improving overall brain blood circulation as well .
further studies currently ongoing in our laboratory should shed further light on the contribution of et-1 via one eta and/or etb receptors and their associated mechanisms , in the etiology of eae in the mouse model .
albeit we show in the present study that mmcp-4 is detrimental in an experimental model of ms , the same concept can not be extended to all types of experimental injuries of the cns .
for example , hendrix and colleagues ( 2013 ) reported the protective role of mast cells ( in part due to mmcp-4 ) in a mouse model of mechanically induced brain injury .
noteworthy , in the same study the authors reversed the protective role of mmcp-4 with a general chymotrypsin inhibitor , chymostatin , which is nonspecific for chymase - dependent processes .
( 2004 ) that , in contrast to spinal cord injury models , eae - induced lesions may facilitate additional levels of axonal reorganization based on the unlesioned fibers of the tract .
these observations support the concept that neuroinflammatory and surgical hemisections of the spinal cord , in the mouse model , alter in opposite fashion the  reserve capacity  of the cns to regenerate .
thus , we suggest that immune - related inflammatory reactions and surgical trauma in the cns prompt opposite roles for mmcp-4 . interestingly , monitoring of inf in our model suggests an enhanced inflammatory reaction in the spinal cord of wt but not mmcp-4 ko mice .
we observed a significantly higher concentration of ifn in the spinal cords of wt compared to mmcp-4 ko mice 2 weeks after immunization .
the decreased inflammatory response is correlated with the observation that mmcp-4 ko mice have decreased proliferation of microglia and astrocytes .
it can nonetheless be explained by decreased responses of cns inflammatory cells in mmcp-4 ko mice which lead to the decreased concentration of proinflammatory molecules , decreased chemotactic gradient , decreased influx of th1 cells , decreased ifn , and , as a result , decreased demyelination . on the other hand ,
preserved integrity of bbb may lead to reduced influx of th1 cells from the periphery , a decreased activation of astrocytes and microglia , a decreased overall inflammation in the cns , and a decreased demyelination .
based on the above observations , the present study reinforces the hypothesis suggested by scandiuzzi et al .
( 2009 ) [ 17 , 18 , 40 ] that mmcp-4 activates cells involved in tissue specific autoimmune reactions .
overall we show in the present study that a single mouse chymase isoform , mmcp-4 , contributes significantly to the etiology and early symptoms associated with a mouse model of ms , namely , eae . if what is reported here in the mouse model can be extended to the clinical situation , it is suggested that targeting chymase rather than the overall mastocytic activity in that particular autoimmune disease will constitute an added value within the currently available therapeutic arsenal against this neurodegenerative disease .
 experimental autoimmune encephalomyelitis ( eae ) is a mouse model that reproduces cardinal signs of clinical , histopathological , and immunological features found in multiple sclerosis ( ms ) . 
 mast cells are suggested to be involved in the main inflammatory phases occurring during eae development , possibly by secreting several autacoids and proteases . among the latter , the chymase mouse mast cell protease 4 ( mmcp-4 ) can contribute to the inflammatory response by producing endothelin-1 ( et-1 ) . 
 the aim of this study was to determine the impact of mmcp-4 on acute inflammatory stages in eae . 
 c57bl/6 wild type ( wt ) or mmcp-4 knockout ( ko ) mice were immunized with mog3555 plus complete freund 's adjuvant followed by pertussis toxin . 
 immunized wt mice presented an initial acute phase characterized by progressive increases in clinical score , which were significantly reduced in mmcp-4 ko mice . 
 in addition , higher levels of spinal myelin were found in mmcp-4 ko as compared with wt mice . 
 finally , whereas eae triggered significant increases in brain levels of mmcp-4 mrna and immunoreactive et-1 in wt mice , the latter peptide was reduced to basal levels in mmcp-4 ko congeners . 
 together , the present study supports a role for mmcp-4 in the early inflammatory phases of the disease in a mouse model of ms . 
1. Introduction
2. Material and Methods
3. Results
4. Discussion