Table Of ContentTakagawaetal.BMCMusculoskeletalDisorders2013,14:40
http://www.biomedcentral.com/1471-2474/14/40
RESEARCH ARTICLE Open Access
Decreased Semaphorin3A expression correlates
with disease activity and histological features of
rheumatoid arthritis
Shu Takagawa1, Fumio Nakamura2*, Ken Kumagai1, Yoji Nagashima3, Yoshio Goshima2 and Tomoyuki Saito1*
Abstract
Background: Rheumatoid arthritis (RA) is an autoimmune disease of which the pathogenetic mechanisms are not
fully understood.Semaphorin3A (Sema3A) has animmuneregulatory role. Neuropilin1 (NRP1), theprimary receptor
for Sema3A, is also a receptor for vascular endothelial growth factor 165(VEGF ). It has been shown that Sema3A
165
competitively antagonizes VEGF165 signaling. Thisstudy investigated whether Sema3A is expressed in synovial
tissues, and is associated with disease activity and thehistologicalfeaturesof synovial tissues from RA patients.
Methods: HumansynovialtissuessampleswereobtainedfromRAandosteoarthritis(OA)patients.Diseaseactivityof
RApatientswascalculatedusingthe28-jointDiseaseActivityScorebasedonC-reactiveprotein(DAS28-CRP).The
histologicalfeaturesofRAsynovialtissueswereevaluatedusingRooney’sinflammationscoringsystem.Thelocalization
ofSema3A,VEGF andNRP1positivecellswasimmunohistochemicallydeterminedinsynovialtissues.Expressionlevels
165
ofSema3A,VEGF-AandNRP1mRNAweredeterminedusingquantitativereal-timepolymerasechainreaction(qPCR).
Results:In OA specimens, Sema3A, VEGF and NRP1 proteins were expressed in the synovial lining and
165
inflammatory cells beneath the lining. Immunohistochemistry revealed the protein expression of Sema3A in
synovial lining cells was decreased in RA tissues compared with OA samples. qPCR analysis demonstrated a
significant reduction of Sema3A mRNA levels in RA synovial tissue samples than in OA and a significant
correlation of the ratio of Sema3A/VEGF-A mRNA expression levels with DAS28-CRP (R = −0.449, p = 0.013).
Sema3A mRNA levels also correlated with Rooney’s inflammation score, especially in perivascular infiltrates of
lymphocytes (R = −0.506, p = 0.004), focal aggregates of lymphocytes (R = −0.501, p = 0.005) and diffuse
infiltrates of lymphocytes (R = −0.536, p = 0.002).
Conclusions: Reduction of Sema3A expression in RA synovial tissues may contribute to pathogenesis of RA.
Keywords: Rheumatoid arthritis, Semaphorin3A, Disease activity score, Histological scoring
Background RA joints, immune cells such as Tand B cells invade the
Rheumatoid arthritis (RA) is a chronic inflammatory dis- hyperplastic synovial membranes [4]. Activated synovial T
ease characterized by progressive joint destruction that ac- andB cellssecrete varioustypesofpro-inflammatorycyto-
companies the proliferation of synovial cells and blood kines including interleukin-1 (IL-1), IL-17 and tumor ne-
vessels as well as invasion of inflammatory cells [1-3]. Al- crosis factor-α (TNF-α). These cytokines induce the
though the initiating factors of RA are unknown, auto- synthesis of matrix degrading enzymes in chondrocytes.
immune reactions are activated in connective tissues. In Synovialfibroblastsalsoproducematrix-degradingenzymes
andcaninvadecartilage,leadingtoitsdestruction[3,4].
Semaphorins are a large family of proteins that func-
*Correspondence:[email protected];[email protected]
2DepartmentofMolecularPharmacologyandNeurobiology,YokohamaCity tion as guidance cues for axonal/dendritic projections.
UniversityGraduateSchoolofMedicine,3-9Fukuura,Kanazawa-ku, Class 3 semaphorins are vertebrate secreted proteins
Yokohama236-0004,Japan
and include seven members, semaphorin3A (Sema3A) to
1DepartmentofOrthopaedicSurgery,YokohamaCityUniversityGraduate
SchoolofMedicine,3-9Fukuura,Kanazawa-ku,Yokohama236-0004,Japan Semaphorin3G (Sema3G) [5]. Sema3A is a repulsive
Fulllistofauthorinformationisavailableattheendofthearticle
©2013Takagawaetal.;licenseeBioMedCentralLtd.ThisisanOpenAccessarticledistributedunderthetermsoftheCreative
CommonsAttributionLicense(http://creativecommons.org/licenses/by/2.0),whichpermitsunrestricteduse,distribution,and
reproductioninanymedium,providedtheoriginalworkisproperlycited.
Takagawaetal.BMCMusculoskeletalDisorders2013,14:40 Page2of11
http://www.biomedcentral.com/1471-2474/14/40
factor for sensory fibers, and Semaphorin3C (Sema3C) Criteria for RA [30] and the American Rheumatism As-
and Semaphorin3F (Sema3F) repulse sympathetic nerve sociation Criteria for OA [31], respectively, as shown in
fibers [6]. Whereas the primary receptor for Sema3A is Table 1. Before arthroplasty, the disease activity of each
neuropilin1 (NRP1), Sema3F has a higher binding affin- RA patient was evaluated using the 28-joint Disease Ac-
ity to neuropilin2 (NRP2). Sema3C binds to both NRP1 tivity Score based on C-reactive protein (DAS28-CRP)
andNRP2[7,8]. [30,32]. This study was approved by the Ethics Commit-
The action of Sema3A is not limited to the nervous tee of Yokohama City University Graduate School of
system as NRP1 is expressed on endothelial cells, kerati- Medicine, and written informed consent was obtained
nocytes, T cells, and tumor cells in breast and prostate from all patients involved in this study (notice of ap-
cancer. Sema3A inhibits angiogenesis, migration of kera- proval Institutional Review Board protocol number:
tinocytes, proliferation of T cells, and migration of B1100513031).
tumor cells [8-11]. In addition, it was recently shown
that Sema3A is involved in the entry of dendritic cells to Hematoxylinandeosin(HE)stainingforhistological
the lymphatic system [12]. Several studies have indicated assessment
that a reduction of Sema3A expression is involved in the Tissue specimens were fixed with 20% formalin and
exacerbation of autoimmune diseases, such as RA and embedded in paraffin. Paraffin sections (4 μm thick)
systemiclupus erythematosus(SLE) [13,14]. were stained with HE. The histological features of RA
NRP1mediatessignaltransductionthroughPlexinAco- synovialtissueswereevaluatedusingRooney’sinflamma-
receptors [15], which are classified into four sub-families, tion scoring system [33]. These histological parameters
PlexinA1–4 [16]. The Sema3A/NRP1/PlexinA complex included the degree of synovial hyperplasia, fibrosis, the
regulatestheactincytoskeletonthroughsmallG-proteins, number of blood vessels, focal and diffuse aggregates of
includingRacandRho[17].Inimmunecells,theRacfam- lymphocytes and perivascular infiltrates of lymphocytes.
ily is associated with the proliferation and activation of B All parameters were scored separately ona scale of0–10
cells [18], and the activation of T cells induced by den- (Table 2). Two investigators (S.T. and Y.N.) independ-
dritic cells [19].NRP1 is alsoa putative markerofregula- ently assessed thehistologicseverity.
tory T cells [20], and therefore Sema3A/NRP1/PlexinA
signalingmaymodulateregulatoryTcellfunctions. Immunohistochemistry
Vascular endothelial growth factor165 (VEGF ), a For immunostaining of Sema3A, antigen retrieval was per-
165
spliced isoform of VEGF-A [21], binds to NRP1 [22]. formed by incubating at 95°C for 30 min in DAKO Target
VEGF is a key regulator of angiogenesis and is involved Retrieval Solution (pH 9.0; DAKO, Glostrup, Denmark).
in the development of inflammation [23]. In RA For immunostaining of NRP1, VEGF , CD3 and CD20,
165
patients, serum VEGF levels positively correlate with the tissue sections were subjected to antigen retrieval by
disease activity score and joint destruction [24,25]. Be- autoclaving in10mMcitratebuffer (pH6.0)for 15minat
cause NRP1 is a common receptor for Sema3A and 121°C.Slideswerethentreatedwith0.3%H O for30min
2 2
VEGF [11,26], the efficacy of VEGF is attributed to to block endogenous peroxidases. Sections for Sema3A,
165 165
Sema3A expression. Indeed, the imbalance between NRP1andVEGF stainingwereblockedwith10%normal
165
Sema3A and VEGF expression levels is also associated goat serum. All sections were incubated at 4°C overnight
with diseaseactivityinseveraltumors[27-29]. with rabbit anti-Sema3A polyclonal antibody (1:200;
These facts prompted us to investigate the possibility Abcam, Cambridge, UK), mouse anti-CD3 monoclonal
that Sema3A expression and/or the balance of Sema3A antibody (1:50; DAKO), mouse anti-CD20 monoclonal
and VEGF expression may regulate the disease activity antibody (DAKO), rabbit anti-NRP1 polyclonal antibody
165
ofRAincludinginflammation,angiogenesisandprolifera- (1:100;SantaCruzBiotechnology,SantaCruz,CA,USA)or
tion of synovial cells. We found that Sema3A expression rabbit anti-VEGF polyclonal antibody (1:100; Millipore,
165
wasdecreasedinRAsynovialtissueswhencomparedwith MA, USA). This was followed by incubation with Envi-
™
osteoarthritis(OA)samples.TheSema3Aexpressionlevel sion , Rabbit/HRP (DAKO) for Sema3A, NRP1 and
™
was also significantly associated with the RA pathological VEGF or Envision /HRP (DAKO) for CD3 and CD20.
165
scoreanddiseaseactivityscore. Immunoreactivity was visualized using 3,3’-diaminobenzi-
dineplus(DAB+,DAKO)forSema3A,NRP1andVEGF ,
165
Methods or 3,3’-diaminobenzidine tetrahydrochloride (DAB; Sigma-
Patientsandsamples Aldrich, St. Louis, MO, USA) for CD3 and CD20. Finally,
Synovial tissue samples were obtained from RA (n = 30) the sections were counterstained with hematoxylin and
and OA (n = 23) patients during total knee arthroplasty. mounted. Controls for Sema3A immunohistochemistry
The diagnosis of patients with RA and OA was based on included preabsorption and co-incubation of the antibody
the revised 1987 American Rheumatism Association withtheantigenpeptide(1μl/ml;Abcam).
Takagawaetal.BMCMusculoskeletalDisorders2013,14:40 Page3of11
http://www.biomedcentral.com/1471-2474/14/40
Table1CharacteristicsofpatientswithOAandRA
OA(n=23) RA(n=30)
Male/female,n 2/21 6/24
Age,mean±SD(range,years) 76.6±5.8(64–84) 69.4±8.1(55–83)
Diseaseduration,mean±SD(years) 10.7±5.1 16.3±9.1
CRP,mean±SD(mg/dl) 0.16±0.30 1.43±1.21
DAS28-CRP,mean±SD – 3.74±0.84
Medications
Prednisolone,n(%) NA 17(56.7)
Methotrexate,n(%) NA 15(50.0)
Sulfasalazine,n(%) NA 7(23.3)
Bucillamine,n(%) NA 6(20.0)
Cyclosporine,n(%) NA 3(10.0)
Actarit,n(%) NA 1(3.3)
TNF-αblockade,n(%) NA 4(13.3)
n,number;OA,osteoarthritis;RA,rheumatoidarthritis;SD,standarddeviation;CRP,C-reactiveprotein;DAS28,28-jointDiseaseActivityScore;NA,notapplicable.
QuantificationofSema3Aimmunostaining time PCR system (Applied Biosystems LLC) based on
Tissuesectionsimmunostainedwithanti-Sema3Aantibody theTaqManW PCRmanufacturer’sprotocol.Theassaywas
wereanalyzedfor12patientswithOAand12patientswith performed in triplicate in optical 96-well reaction plates
RA. The total immunostaining intensity in the lining layer covered with optical adhesive cover in a volume of 10 μl
was measured using a BZ-9000 microscope (Keyence, containing 0.5 μl Taqman Gene Expression Assay 20X for
Osaka, Japan) equipped with Dynamic Cell count software human Sema3A (assay ID Hs01085496_m1, GenBank ac-
BZ-H1C(Keyence).Immunostainingintensityperunitwas cession number NM_006080, Applied Biosystems LLC),
calculatedasdescribedpreviously[34]. VEGF-A (assay ID Hs00173626_m1, GenBank accession
number NG_008732, Applied Biosystems LLC), NRP1
Quantitativereal-timepolymerasechainreaction(qPCR) (assay ID Hs00826128_m1, GenBank accession number
Total RNA was extracted from synovial tissues using an NM_003873; Applied Biosystems LLC) and β-actin (assay
Illustra RNA spin Mini Kit (GE Healthcare, Bucking- ID 4326315E, GenBank accession number NM_001101;
hamshire, UK) according to the manufacturer’s instruc- Applied Biosystems LLC), 5 μl Taqman Fast Advanced
tions. RNA was reversed transcribed into cDNA using a MasterMix2X,2μlcDNAtemplateand2.5μlRNase-free
PrimeScript RT Reagent Kit (Takara Bio, Ohtsu, Japan). water. The default ABI 7900HT amplification conditions
The cDNA synthesizedfrom 1μgoftotal RNAwasused were20secat95°C,followedby1secat95°Cand20secat
as the template in each reaction. qPCR analysis was per- 60°C for 40 cycles. A standard curve, derived from known
formed using an Applied Biosystems 7900HT Fast Real- serial dilutions of RA synovial tissue, was constructed to
Table2Rooney’sinflammationscoringsystemforpatientswithrheumatoidarthritis
Score
Histologicfeature 0 1 2 3 4 5 6 7 8 9 10
Synoviocytehyperplasiaa 1 2 3 4 5 6 7 8 9 10 >10
Fibrosisb <10 <15 <20 <25 <30 <40 <50 <60 <70 <80 ≥80
Proliferatingbloodvesselsc 0–3 4–5 6–7 8–9 10–11 12–13 14–15 16–17 18–19 20–22 >22
Perivascularinfiltratesoflymphocytesd <5 10 20 30 40 50 60 70 80 90 100
Focalaggregatesoflymphocytese <11 15 20 25 30 35 40 45 50 55 >55
Diffuseinfiltratesoflymphocytesf 0 10 20 30 40 50 60 70 80 90 100
aAnormalsynoviocytemonolayerwasscoredas0.Thescorewasincreasedasthesynoviocytelininglayerincreasedindepth.bSectionscontaining<10%fibrous
tissueinthesublininglayerswerescoredas0.Thescoreincreasedasthepercentageoffibrosisinthesectionincreased.cThreeorfewervesselsperhighpower
field(HPF)werescoredas0.ThescoreincreasedasthenumberofvesselsperHPFincreased.dWhennolymphocyteswereobservedaroundvessels,itwasscored
as0.Thescoreincreasedasthepercentageofvesselssurroundedbylymphocytesincreased.Subsequently,thediameteroftheperivascularlymphocyteswas
gradedasmild=2–4cellsindiameter,moderate=5–7cellsindiameter,andsevere=8–10cellsindiameter.Wheretheperivascularlymphocyteswere
consideredmildorsevere,theoriginalscorewasloweredby1pointorraisedby1point,respectively.eAbsenceoffocalaggregatesoflymphocytes,which
exceeded10cellsindiameter,werescoredas0.Thescoreincreasedasthecellnumbersinthediameterofthefocalaggregatesincreased.fAnestimatewas
madeofthepercentageofcellsperHPFthatwerelymphocytes.Thescoreincreasedasthepercentageoflymphocytesincreased.
Takagawaetal.BMCMusculoskeletalDisorders2013,14:40 Page4of11
http://www.biomedcentral.com/1471-2474/14/40
calculatearbitraryvaluesofmRNAlevelsandtocorrectfor thehyperplasticlininglayerwaslowerinRAtissuescom-
differences in primer efficiencies. The obtained data were pared with OA samples (Figure 1E). Additionally, numer-
standardizedusingthereferencegene,β-actin. ous inflammatory cells in the sublining layer expressed
Sema3A. In OA and RA synovial tissues, VEGF was
165
Double-stainingImmunofluorescence expressedinthelininglayerandintheinflammatorycells
Synovial tissue specimens embedded in Optimum Cut- ofthesublininglayer (Figure1G,J).TheVEGF expres-
165
ting Temperature compound (Sakura Finetek Japan, sion density in the lining layer did not differ between RA
Tokyo, Japan) were sectioned (5 μm thick). The sections and OA samples. NRP1, the shared receptor for Sema3A
were then fixed in cold acetone for 5 min at 4°C and and VEGF , wasexpressed in synovialcellsof thelining
165
rinsed in phosphate buffered saline (PBS). To eliminate layer,alongwithinflammatorycellsandvascularendothe-
nonspecific protein binding, the samples were incubated lialcellsinthesublininglayer.TheNRP1expressiondens-
with 10% normal goat serum for 30 min at room ity in the lininglayerofRA wasnot significantlydifferent
temperature. The samples were incubated with rabbit tothatofOA(Figure1H,K).ThelocalizationofSema3A,
anti-NRP1 polyclonal antibody (1:100; Santa Cruz Bio- VEGF , and NRP1 almost overlapped, suggesting that
165
technology) and mouse anti-CD20 monoclonal antibody functional competition for Sema3A and VEGF may in-
165
(DAKO) overnight at 4°C. This was followed by incuba- fluencethese cells. Toevaluate theinfiltrationofimmune
tion with Alexa 488-labeled goat anti-rabbit antibody cells, sections were stained with anti-CD3 antibody, a
(Applied Biosystems LLC, Foster City, CA, USA) and markerforTcellsandanti-CD20antibody,amarkerforB
Alexa 594-labeled goat anti-mouse antibody (Applied cells. While T cells (CD3) were sporadically localized in
BiosystemsLLC)for40 minat37°C.Finally,the sections the sublining layer of OA specimens, the number of T
were mounted with aqueous mounting medium. The cells was greater in RA synovial tissues (Figure 1I, L). B
distributions were analyzed by confocal microscopy cellswerealsosporadicallylocalizedinthesublininglayer
using a Zeiss LSM510 confocal laser microscope (Carl of OA specimens and large numbers of B cells were
Zeiss,Oberkochen,Germany). observed in the lymphoid follicles of RA synovial tissues
(Figure 1M, N). We quantified Sema3A-immunostaining
Statisticalanalysis signal in the lining layer of RA and OA specimens.
Statistical analyses were performed using SPSS 11.0 for Sema3A-immunostaining intensity per unit area of lining
Windows(SPSSInc,Chicago,IL).TheMann–WhitneyU- layer was significantly less in RA patients compared with
test and Spearman’s rank correlation coefficient were used OA(Figure1O).
totestthedifferences.Apvalue<0.05wasconsideredsig-
nificant. We calculated a posterior power of this study ExpressionofSema3A,VEGF-AandNRP1mRNAand
using G*Power (Faul, Erdfelder, Lang, & Buchner, 2007). correlationwithDAS28-CRPinRA
Allstatisticalpowersinthisstudyweregreaterthan80%. To confirm changes in Sema3A expression in RA, we
examined mRNA expression levels in OA and RA joint
Results specimens using qPCR analysis. To evaluate the expres-
Sema3A,VEGF165,NRP1andCD3expressioninOAand sion levels of VEGF165 we used VEGF-A primers in the
RAsynovialtissues qPCR experiments. Sema3A mRNA levels in synovial
To investigate the involvement of Sema3A in RA patho- tissue samples were significantly lower in RA (mean ex-
genesis, we performed immunohistochemical staining pression level 1.80) than in OA (mean expression level
for Sema3A, VEGF and NRP1 expression in serial 6.68; p < 0.0001; Figure 2A). The mRNA expression of
165
synovial serial sections from RA and OA patients. HE VEGF-A or NRP1 was not significantly different between
staining demonstrated that synovial tissues from OA RAandOA(Figure2B,C).Wealsoobtainedsimilarresults
patients contained two layers, the lining and sublining withVEGF primer in preliminary study (Additional file
165
layers (Figure 1A). Sema3A was mainly expressed in the 1: Figure S1A). We next examined the correlation of
lining layer and a small number of inflammatory cells Sema3A expression levels with DAS28-CRP, the disease
were present in the sublining layer of OA samples activity score for RA. A negative correlation was observed
(Figure 1B). This immunoreactive signal was abolished between Sema3A expression levels and DAS28-CRP (R =
by the preincubation of anti-Sema3A antibody with the −0.409, p = 0.025; Figure 2D). This suggested that the
antigen peptide (Figure 1C), confirming the specificity of reduction of Sema3A expression might augment the
Sema3A immunostaining. In RA specimens, there was a disease activity of RA. VEGF-A expression levels did
marked increase in synovial tissue thickness of the lining not significantly correlate with DAS28-CRP (R = 0.198,
layer caused by hyperplasia of synovial cells and numer- p = 0.295; Figure 2E). We also found a negative correl-
ous infiltrating inflammatory cells in the sublining layer ation between Sema3A/VEGF-A ratios and DAS28-CRP
(Figure 1D). The immunostaining signal of Sema3A in (R= −0.449, p = 0.013; Figure 2F).
Takagawaetal.BMCMusculoskeletalDisorders2013,14:40 Page5of11
http://www.biomedcentral.com/1471-2474/14/40
Figure1Histological/immunohistochemicalanalysisofSema3A,NRP1,VEGF andCD3inOAandRAsynovialtissue.Representative
165
HEstainingofOA(A)andRA(D)synovialtissues.OAsynovialtissuescontainlining(LL)andsublining(SL)layers.RAsynovialtissuesaremarked
bythehyperplasiaofsynovialtissuesinthelininglayerandnumerousinfiltratedinflammatorycells(IC)inthesublininglayer.Sema3Aexpression
wasdetectedinthelininglayerandinflammatorycellsinthesublininglayerofOA(B)andRA(E)synovialtissues.ThedensityoftheSema3A
signalinthelininglayerwaslowerinRAthanOA.Peptide-neutralizedanti-Sema3AantibodiesdidnotstaintissuesectionsfromOA(C)andRA
(F).NRP1andVEGF localizedtothesameareasasSema3AinOA.VEGF expressioninthelininglayerinRAtissueswassimilarwithOA
165 165
(G,J).TheNRP1expressionlevelinthelininglayerofRAwassimilartoOA(H,K).Tcells(CD3)andBcells(CD20)weredetectedamong
inflammatorycellsinthesublininglayerofOAandRAsynovialtissues(I,L,M,N).ThenumbersofTcellsandBcellswerehigherinRAsynovial
tissuescomparedwithOA.Sectionswerecounterstainedwithhematoxylin.Scalebars=50μminthewholeimageviewand25μminthe
magnifiedview.ImmunostainingoflininglayerSema3AwassignificantlydecreasedinRA(n=12)synovialtissuescomparedwithOA(n=12)
subjects(O).ResultsarepresentedasrelativevaluescomparedwithOAsubjects.Theboxplotsdemonstratethe10thand90thpercentile
(whiskers),the25thand75thpercentile,andthemedian.PvalueswereobtainedusingtheMann–WhitneyU-test.
Takagawaetal.BMCMusculoskeletalDisorders2013,14:40 Page6of11
http://www.biomedcentral.com/1471-2474/14/40
Figure2ExpressionofSema3A,VEGF-AandNRP1mRNAinsynovialtissues.ExpressionlevelsofSema3A,VEGF-AandNRP1mRNAwere
measuredusingreal-timePCR.ThemRNAlevelswerenormalizedtotheexpressionofβ-actin.Theboxplotsdemonstratethe10thand90th
percentile(whiskers),the25thand75thpercentile,andthemedian.Sema3AmRNAlevelsweresignificantlydecreasedinRA(n=30)synovial
tissuescomparedwithOA(n=23)(A).VEGF-AandNRP1mRNAlevelswerenotsignificantlyaltered(B,C).CorrelationofSema3AmRNAlevels
andDAS28-CRP(D);VEGF-AmRNAlevelsandDAS28-CRP(E);andSema3A/VEGF-AmRNAlevelsandDAS28-CRP(F).Pvalueswereobtainedusing
theMann–WhitneyU-test.wereexaminedusingSpearman’srankcorrelationcoefficient.
CorrelationofSema3AmRNAlevelswithhistological NRP1-positive inflammatory cells, we stained sequential
featuresinRAsynovialtissues tissuesectionswithmarkersforTcells(CD3)andBcells
To confirm the inverse correlation of Sema3A mRNA (CD20). NRP1 was abundantly expressed in the inflam-
expression levels and RA disease activity, we compared matory cells of lymphoid follicles (Figure 4A). In RA
the relative Sema3A mRNA expression levels with histo- synovium,Tcells, B cells and dendritic cells [35] can be
logical parameters of RA synovial tissues. This included arranged in sophisticated organizations that resemble
the degree of synovial hyperplasia, fibrosis, number of the microstructures usually formed in secondary lymph-
blood vessels present, perivascular infiltrates of lympho- oid organs. Interestingly, NRP1-expressing dendritic
cytes, focal aggregates of lymphocytes and diffuse infil- cells also exist in the lymphoid follicle/lymphocyte focal
trates of lymphocytes. No significant correlation aggregates. Immunohistochemical staining of sequential
between Sema3A expression and synovial hyperplasia, fi- slices from the lymphoid follicles showed that NRP1-
brosis or the number of blood vessels was observed positive cells also expressed CD3 or CD20 (Figure4B, C).
(Figure 3A–C). In contrast, perivascular infiltrates of Since lymphoid follicles were mainly composed of B cells
lymphocytes, focal aggregates of lymphocytes and diffuse [36], we also performed double immunofluorescence
infiltrates of lymphocytes significantly correlated with staining of NRP1 and CD20. The NRP1 positive signal
Sema3AmRNA expression levels (R =−0.506, p= 0.004; colocalizedwiththeCD20positivesignalintheinflamma-
R = −0.501, p = 0.005; R = −0.536, p=0.002 respectively; torycellsoflymphocyteaggregates(Figure4D-F).
Figure 3D–F). These results indicated that Sema3A
mRNA reduction was mainly associated with immune Discussion
reactionsinthesynovial tissuefrom RApatients. It has been suggested that abnormalities of the immune
system play an important role in the pathogenesis of
ExpressionofNRP1inCD20-positiveBcellsin chronic inflammatory conditions such as RA. This study
lymphocyteaggregates revealed Sema3A expression levels significantly corre-
Histological scores and Sema3A expression levels latedwiththehistologicalscoreofperivascularinfiltrates
revealed that a decrease in Sema3A augmented focal of lymphocytes, focal aggregates of lymphocytes and dif-
aggregates of lymphocytes. To examine the cell types of fuse infiltrates of lymphocytes. Sema3A is involved in
Takagawaetal.BMCMusculoskeletalDisorders2013,14:40 Page7of11
http://www.biomedcentral.com/1471-2474/14/40
Figure3ExpressionlevelsofSema3AmRNAcorrelatewithhistologicalfeaturesinRAsynovialtissues.CorrelationsbetweenSema3A
mRNAexpressionlevelsandhistologicalparametersofRAsynovialtissueswereassessed.Histologicalparameterswerescoredseparatelyona
scaleof0–10accordingtoRooney’sinflammationscoringsystem.CorrelationofSema3AmRNAlevelsandsynoviocytehyperplasia(A);Sema3A
mRNAlevelsandfibrosis(B);Sema3AmRNAlevelsandproliferatingbloodvessels(C);Sema3AmRNAlevelsandperivascularinfiltratesof
lymphocytes(D);Sema3AmRNAlevelsandfocalaggregatesoflymphocytes(E);andSema3AmRNAlevelsanddiffuseinfiltratesoflymphocytes
(F).CorrelationswereexaminedusingSpearman’srankcorrelationcoefficient.
Figure4Immunohistochemicalanddoubleimmunofluorescencestainingoflymphocyteaggregates.NRP1wasabundantlyexpressedby
inflammatorycellsoflymphocytesaggregates(A).ImmunohistochemicalstainingofsequentialsectionsshowedthatNRP1-positivecellsin
lymphocytesaggregatesalsoexpressedCD3(B)orCD20(C).MicrographsshowthedoubleimmunofluorescencedetectionofNRP1(D,green)
andCD20(E,red)withthemergedimages(F).TheNRP1positivesignalcolocalizedwiththeCD20positivesignalintheinflammatorycellsof
lymphocyteaggregates.A–C,scalebars=50μm;D–F,scalebars=10μm.
Takagawaetal.BMCMusculoskeletalDisorders2013,14:40 Page8of11
http://www.biomedcentral.com/1471-2474/14/40
immune responses, and its expression levels are altered that the presence of calcium and histamine could modu-
in several autoimmune diseases [29,37,38]. The NRP1 lateSema3Aexpressionlevelsinhumankeratinocytesand
and PlexinA complex mediates Sema3A signaling in the fibroblasts [43]. Since histamine is associated with RA
immune system [39]. NRP1 is expressed by regulatory T pathogenesis [44,45], it may be responsible for the
cells, a subset of T cells [40] and PlexinA4 regulates T decreasedSema3AlevelsobservedinRAsynovialtissues.
cell proliferation and activation through inhibition of the We found that mRNA expression of VEGF-A was not
actin-cytoskeleton rearrangement and T cell receptor significantly altered in OA and RA synovial tissues.
polarization [9]. Recently, it was reported that Sema3A Hashimoto et al. reported staining intensity for VEGF
promotes regulatory Tcells by enhancing IL-10 produc- expression did not differ between RA and OA synovial
tion [14]. Regulatory T cells play an important role in lining [46] and Lowin et al. reported that VEGF ex-
165
maintaining immunological self-toleranceby suppressing pression did not differ in the chronically inflamed tissue
autoreactive T cells [41]. Immunohistochemical staining of RA patients and OA patients [47]. In contrast, several
in the current study revealed that Sema3A is expressed reports showed altered expression of VEGF in RA. Lee
in the lining layer. Thus, in OA joints, secreted Sema3A et al. observed significantly higher levels of VEGF pro-
fromthelininglayermayenhanceregulatoryTcellfunc- tein in RA compared with OA synovial fluid and serum
tions to suppress autoimmune responses in the sublining [48]. Kurosawa et al. observed significant correlations of
layer. In RA, the reduction of Sema3A may abrogate the serum VEGF levels with DAS28-CRP scores [24]. The
functions of regulatory Tcells, thus allowing the infiltra- elevation of VEGF protein in RA synovial fluid and
tion and focal aggregation of autoreactive lymphocytes in serum may explain the higher total number of VEGF-
thesublininglayer.Furtherinvestigationofthecorrelation producing cells in the region [46]. Indeed, our study
between Sema3A and IL-10 expression levels may in- showed nosignification correlationbetween VEGF-A ex-
creaseourunderstandingoftheirrolesinRA. pression levels and DAS28-CRP scores (Figure 2), but
Catalano reported synovial tissues derived from healthy there was a marked increase of synovial tissue thickness
controls,OAandRApatientsexhibitednosignificantdif- of the lining layer in RA (Figure 1). Thus, VEGF levels
ferences although the relative Sema3A expression was in RA serum may be increased as previously reported
lowest in RA samples [14]. This discrepancy might be [24]. In an earlier study, Ikeda et al. reported VEGF165
explained by the difference in sampling numbers, which wasexpressedin41%ofRAsamples(17patients)butnot
can influence statistical power, and/or by different qPCR in OA samples (8 patients) using reverse transcription-
methods used. We quantified Sema3A mRNA expression PCR [49]. They also found NRP1 was up-regulated in RA
in a larger number of RA patients (n = 30) and OA synovial tissues. However, we did not observe significant
patients (n = 23), whereas Catalano used relatively small alterations of VEGF-A (Figure 2B) or NRP1 (Figure 2C)
numbersofRA(n=10),OA(n=10)patientsandhealthy between OA and RA specimens. Kim et al. reported that
controls (n = 5). For qPCR, we used the standard curve NRP1expressionwassimilarinOAand RAusingimmu-
method for relative quantification, whereas Catalano used nohistochemical analyses [50]. These discrepancies might
acomparativeCtmethod. be explained by the difference in sampling numbers, dis-
SequentialimmunohistochemicalstainingrevealedthatB easedurationofthepopulationsstudied,extentofinflam-
cells were observed mainly in the lymphoid follicles, con- mation, use of anti-rheumatoid drugs, and/or by different
sistent with a previous report [36]. Here we observed that methodsforanalysisofNRP1expressionlevels.Additional
B cells in the follicles expressed NRP1 (Figure 4). Vadasz investigations in a large sample considering several differ-
et al. recently suggested that Sema3A can modulate the entconditionsarerequiredtoclarifythesediscrepancies.
autoimmune properties of B cells in SLE [13]. Thus, Although Sema3A inhibits endothelial formation and
Sema3A may exert similar functions in NRP1-positive B angiogenesis due to competition withVEGF [51], this
165
cells from RA synovial tissues. Vadasz et al. also reported study did not identify a significant correlation between
Sema3A serum levels were significantly lower in SLE Sema3A mRNA expression levels and blood vessel dens-
patients(p<0.0001)andRApatients(p=0.047)compared ity in RA synovial tissues. This suggests that angiogen-
withhealthycontrols[13].SinceSema3Aisa secretedsol- esis in RA lesions may be regulated by other mediators,
ubleproteinandcanenterthesystemiccirculation[16,42], such as placenta growth factor 1, IL-2 and hepatocyte
lower Sema3A serum levels in RA patients may reflect its growth factor[47,52].
reduced expression in knee joints and/or other organs. The imbalance between Sema3A and VEGF may also
Additional investigations to quantify the serum levels of affect the etiology of RA. Although VEGF-A expression
Sema3A from OA and RA patients may contribute to fur- levelsdidnotexhibitasignificantcorrelationwithDAS28-
therelucidationofpathogenesisofthedisease. CRP scores, the Sema3A/VEGF-A mRNA ratios demon-
It is unclear which factors regulate Sema3A expression strated a relationship with the RA clinical score. Several
in synovial tissues. However, Fukamachi et al. reported studies have demonstrated that anti-NRP1 peptides
Takagawaetal.BMCMusculoskeletalDisorders2013,14:40 Page9of11
http://www.biomedcentral.com/1471-2474/14/40
suppressed the survival, adhesion and migration of Additional file
VEGF -inducedsynovialcells,whichcontributetocartil-
165
age destruction in RA [50,53]. VEGF165 also increased the Additionalfile1:ExpressionofVEGF165mRNAinsynovialtissues:
preliminarystudy.ExpressionlevelsofVEGF mRNAweremeasured
productionofcytokinesbyhumanperipheralbloodmono- 165
byreal-timePCR.ThemRNAlevelswerenormalizedtotheexpressionof
nuclear cells [54]. Sema3A may inhibit the action of β-actin.Theboxplotsdemonstratethe10thand90thpercentile
VEGF165onsynovialcellsandinflammatorycellsinacom- (whiskers),the25thand75thpercentile,andthemedian.VEGF165mRNA
levelswerenotsignificantlyalteredinRA(n=25)synovialtissues
petitivemanner.
comparedwithOA(n=17)(A).VEGF expressionlevelsdidnot
Innervation is important in the pathogenesis of arth- significantlycorrelatewithDAS28-CRP16(5B).
ritis.Primaryafferent sensory nervefibers areproinflam-
matory, whereas sympathetic nerve fibers are anti-
Abbreviations
inflammatory [55]. In RA synovial tissues, numbers of DAS28-CRP:28-jointdiseaseactivityscorebasedonc-reactiveprotein;
sympathetic nerve fibers decreased while sensory nerves HE:Hematoxylinandeosin;IL:Interleukin;NRP1:Neuropilin1;
NRP2:Neuropilin2;OA:osteoarthritis;qPCR:Quantitativereal-time
increased when compared with OA [56]. The loss of
polymerasechainreaction;RA:Rheumatoidarthritis;Sema3A:Semaphorin3A;
sympathetic nerve fibers in RA may be caused by Sema3C:Semaphorin3C;Sema3F:Semaphorin3F;Sema3G:Semaphorin3G;
increased Sema3C and its soluble receptor NRP2 [6,55]. SD:Standarddeviation;SLE:Systemiclupuserythematosus;TNF-α:Tumor
necrosisfactor-α;VEGF :Vascularendothelialgrowthfactor165.
However, the mechanism of increased sensory nerve 165
fibers in RA synovial tissues is not fully understood.
Competinginterests
Decreased Sema3A expression in RA may facilitate the Theauthorsdeclarethattheyhavenocompetinginterests.Thisworkwas
increase of sensory nerves over sympathetic nerves in supportedbysupportedbyGrants-in-AidforScientificResearchnos.
30170517(toT.S)andnos.23590429(toY.N)fromtheMinistryofEducation,
inflamedRA synovium [55].
Culture,Sports,ScienceandTechnologyofJapan(MEXT),andbyGrantnos.
Several limitations of our study should be addressed. 07085023(toY.G.)fromtheNationalProjectonProteinStructuraland
First, this study population may be influenced by envir- FunctionalAnalysesofMEXT.
onmental factors as well as genetic factors. Second,
Authors’contributions
patients enrolled in our study were in the advanced
TSparticipatedinthedesignofthestudy,performedtheexperimentsand
stages of RA disease (mean duration 16.3 years). Thus, statisticalanalysis,draftedandrevisedthemanuscript.NFdesignedthe
our data do not reflect the pathogenesis of RA during study,analyzeddata,draftedandrevisedthemanuscriptcritically.KKhelped
withcollectionandacquisitionofthedataandthesynoviumandrevised
the early stages. Third, anti-rheumatoid drugs may
themanuscriptcritically.NYassistedinscoringhistologicalparametersand
alter the expression level of Sema3A and other mole- revisedthemanuscriptcritically.GYdesignedthestudyandrevisedthe
cules. In this study 12 patients (40%) received metho- manuscriptcritically.SThelpedwithcollectionoftheclinicalmaterialsand
revisedthemanuscriptcritically.Allauthorsreadandapprovedthefinal
trexate (MTX) (mean prednisolone daily dose 2.9 mg), 4
manuscriptforpublication.
patients (13.3%) received TNF-α blockade (3 patients in
combination with MTX, 1 patient in combination with Acknowledgments
sulfasalazine, mean prednisolone daily dose 3.6 mg), 11 TheauthorswishtothankMs.TamiyoTaniguchi,Mrs.AtsukoFukuiandMrs.
InadaMaefortheirtechnicalandsecretarialassistance.
patients received Disease Modifying Antirheumatic
Drugs (DMARDs) without MTX (mean prednisolone Authordetails
daily dose 1.6 mg), and 3 patients received prednisolone 1DepartmentofOrthopaedicSurgery,YokohamaCityUniversityGraduate
SchoolofMedicine,3-9Fukuura,Kanazawa-ku,Yokohama236-0004,Japan.
alone(dailydose6.2mg).Althoughtherewasnostatisti-
2DepartmentofMolecularPharmacologyandNeurobiology,YokohamaCity
cally significant difference in Sema3A expression, UniversityGraduateSchoolofMedicine,3-9Fukuura,Kanazawa-ku,
DAS28-CRP, or Rooney’s inflammation score between Yokohama236-0004,Japan.3DepartmentofMolecularPathology,Yokohama
the MTX–treated group and other groups (data not CityUniversityGraduateSchoolofMedicine,3-9Fukuura,Kanazawa-ku,
Yokohama236-0004,Japan.
shown), we cannot rule out the possibility of altered
Sema3A expression by anti-rheumatoid drugs, because Received:10September2012Accepted:12January2013
Published:23January2013
eachgroup samplesizeissmall inthisstudy.
References
Conclusions 1. NagaokaA,TakizawaN,TakeuchiR,InabaY,SaitoI,NagashimaY,SaitoT,
This study demonstrated that Sema3A expression levels AokiI:PossibleinvolvementofpeptidylprolylisomerasePin1in
rheumatoidarthritis.PatholInt2011,61(2):59–66.
and the disease activity score in patients with RA nega-
2. MatsuoK,AritoM,NoyoriK,NakamuraH,KurokawaMS,MasukoK,
tively correlated with histological parameters in RA syn- OkamotoK,NagaiK,SuematsuN,YudohK,etal:Arthritogenicityof
ovial tissues. This suggests that Sema3A may have a annexinVIIrevealedbyphosphoproteomicsofrheumatoidsynoviocytes.
AnnRheumDis2011,70(8):1489–1495.
suppressive effect on the pathological severity of RA.
3. NeumannE,LefevreS,ZimmermannB,GayS,Muller-LadnerU:
Further elucidation of the role of Sema3A in joints may Rheumatoidarthritisprogressionmediatedbyactivatedsynovial
elucidate the biological and pathological functions of fibroblasts.TrendsMolMed2010,16(10):458–468.
4. McInnesIB,SchettG:Cytokinesinthepathogenesisofrheumatoid
Sema3A andallow thedevelopment ofnewstrategiesfor arthritis.NatRevImmunol2007,7(6):429–442.
thetreatmentofRA. 5. YazdaniU,TermanJR:Thesemaphorins.GenomeBiol2006,7(3):211.
Takagawaetal.BMCMusculoskeletalDisorders2013,14:40 Page10of11
http://www.biomedcentral.com/1471-2474/14/40
6. FassoldA,FalkW,AndersS,HirschT,MirskyVM,StraubRH:Soluble expressionofappropriatesemaphorinreceptorsbytumorcells.PLoS
neuropilin-2,anerverepellentreceptor,isincreasedinrheumatoid One2008,3(9):e3287.
arthritissynoviumandaggravatessympatheticfiberrepulsionand 27. BarresiV,TuccariG:Increasedratioofvascularendothelialgrowthfactor
arthritis.ArthritisRheum2009,60(10):2892–2901. tosemaphorin3Aisanegativeprognosticfactorinhuman
7. KolodkinAL,LevengoodDV,RoweEG,TaiYT,GigerRJ,GintyDD: meningiomas.Neuropathology2010,30(5):537–546.
NeuropilinisasemaphorinIIIreceptor.Cell1997,90(4):753–762. 28. OsadaR,HoriuchiA,KikuchiN,OhiraS,OtaM,KatsuyamaY,KonishiI:
8. GaurP,BielenbergDR,SamuelS,BoseD,ZhouY,GrayMJ,DallasNA,FanF, Expressionofsemaphorins,vascularendothelialgrowthfactor,andtheir
XiaL,LuJ,etal:Roleofclass3semaphorinsandtheirreceptorsintumor commonreceptorneuropilinsandalleiclossofsemaphorinlocusin
growthandangiogenesis.ClinCancerRes2009,15(22):6763–6770. epithelialovarianneoplasms:increasedratioofvascularendothelial
9. LepelletierY,MouraIC,Hadj-SlimaneR,RenandA,FiorentinoS,BaudeC, growthfactortosemaphorinisapoorprognosticfactorinovarian
ShirvanA,BarzilaiA,HermineO:Immunosuppressiveroleofsemaphorin- carcinomas.HumPathol2006,37(11):1414–1425.
3AonTcellproliferationismediatedbyinhibitionofactincytoskeleton 29. BachelderRE,LipscombEA,LinX,WendtMA,ChadbornNH,EickholtBJ,
reorganization.EurJImmunol2006,36(7):1782–1793. MercurioAM:Competingautocrinepathwaysinvolvingalternative
10. KurschatP,BielenbergD,Rossignol-TallandierM,StahlA,KlagsbrunM: neuropilin-1ligandsregulatechemotaxisofcarcinomacells.CancerRes
NeuronrestrictivesilencerfactorNRSF/RESTisatranscriptionalrepressor 2003,63(17):5230–5233.
ofneuropilin-1anddiminishestheabilityofsemaphorin3Atoinhibit 30. ArnettFC,EdworthySM,BlochDA,McShaneDJ,FriesJF,CooperNS,Healey
keratinocytemigration.JBiolChem2006,281(5):2721–2729. LA,KaplanSR,LiangMH,LuthraHS,etal:TheAmericanrheumatism
11. MiaoHQ,SokerS,FeinerL,AlonsoJL,RaperJA,KlagsbrunM:Neuropilin-1 association1987revisedcriteriafortheclassificationofrheumatoid
mediatescollapsin-1/semaphorinIIIinhibitionofendothelialcellmotility: arthritis.ArthritisRheum1988,31(3):315–324.
functionalcompetitionofcollapsin-1andvascularendothelialgrowth 31. AltmanR,AschE,BlochD,BoleG,BorensteinD,BrandtK,ChristyW,Cooke
factor-165.JCellBiol1999,146(1):233–242. TD,GreenwaldR,HochbergM,etal:Developmentofcriteriaforthe
12. TakamatsuH,TakegaharaN,NakagawaY,TomuraM,TaniguchiM,Friedel classificationandreportingofosteoarthritisl.Classificationof
RH,RayburnH,Tessier-LavigneM,YoshidaY,OkunoT,etal:Semaphorins osteoarthritisoftheknee.Diagnosticandtherapeuticcriteriacommittee
guidetheentryofdendriticcellsintothelymphaticsbyactivating oftheAmericanrheumatismassociation.ArthritisRheum1986,
myosinII.NatImmunol2010,11(7):594–600. 29(8):1039–1049.
13. VadaszZ,HajT,HalaszK,RosnerI,SlobodinG,AttiasD,KesselA,KesslerO, 32. InoueE,YamanakaH,HaraM,TomatsuT,KamataniN:Comparisonof
NeufeldG,ToubiE:Semaphorin3Aisamarkerfordiseaseactivityanda diseaseactivityscore(DAS)28-erythrocytesedimentationrateand
potentialimmunoregulatorinsystemiclupuserythematosus.ArthritisRes DAS28-C-reactiveproteinthresholdvalues.AnnRheumDis2007,
Ther2012,14(3):R146. 66(3):407–409.
14. CatalanoA:Theneuroimmunesemaphorin-3Areducesinflammationand 33. RooneyM,CondellD,QuinlanW,DalyL,WhelanA,FeigheryC,BresnihanB:
progressionofexperimentalautoimmunearthritis.JImmunol2010, AnalysisoftheHistologicvariationofsynovitisinrheumatoidarthritis.
185(10):6373–6383. ArthritisRheum1988,31(8):956–963.
15. GoshimaY,ItoT,SasakiY,NakamuraF:Semaphorinsassignalsforcell 34. KouK,NakamuraF,AiharaM,ChenH,SetoK,Komori-YamaguchiJ,
repulsionandinvasion.JClinInvest2002,109(8):993–998. KambaraT,NagashimaY,GoshimaY,IkezawaZ:Decreasedexpressionof
16. TakahashiT,FournierA,NakamuraF,WangLH,MurakamiY,KalbRG, semaphorin-3A,aneurite-collapsingfactor,isassociatedwithitchin
FujisawaH,StrittmatterSM:Plexin-neuropilin-1complexesformfunctional psoriaticskin.ActaDermVenereol2012,92(5):521–528.
semaphorin-3Areceptors.Cell1999,99(1):59–69. 35. TakemuraS,BraunA,CrowsonC,KurtinPJ,CofieldRH,O'FallonWM,
17. SasakiY,ChengC,UchidaY,NakajimaO,OhshimaT,YagiT,TaniguchiM, GoronzyJJ,WeyandCM:Lymphoidneogenesisinrheumatoidsynovitis.J
NakayamaT,KishidaR,KudoY,etal:FynandCdk5mediatesemaphorin- Immunol2001,167(2):1072–1080.
3Asignaling,whichisinvolvedinregulationofdendriteorientationin 36. SakuraiN,KuroiwaT,IkeuchiH,HiramatsuN,MaeshimaA,KanekoY,
cerebralcortex.Neuron2002,35(5):907–920. HiromuraK,NojimaY:ExpressionofIL-19anditsreceptorsinRA:
18. WalmsleyMJ,OoiSK,ReynoldsLF,SmithSH,RufS,MathiotA,VanesL, potentialroleforsynovialhyperplasiaformation.Rheumatology(Oxford)
WilliamsDA,CancroMP,TybulewiczVL:CriticalrolesforRac1andRac2 2008,47(6):815–820.
GTPasesinBcelldevelopmentandsignaling.Science2003, 37. KumanogohA,KikutaniH:Semaphorinsandtheirreceptors:novel
302(5644):459–462. featuresofneuralguidancemolecules.ProcJpnAcadSerBPhysBiolSci
19. BenvenutiF,HuguesS,WalmsleyM,RufS,FetlerL,PopoffM,Tybulewicz 2010,86(6):611–620.
VL,AmigorenaS:RequirementofRac1andRac2expressionbymature 38. TominagaM,OgawaH,TakamoriK:Decreasedproductionofsemaphorin
dendriticcellsforTcellpriming.Science2004,305(5687):1150–1153. 3Ainthelesionalskinofatopicdermatitis.BrJDermatol2008,
20. BruderD,Probst-KepperM,WestendorfAM,GeffersR,BeissertS,LoserK, 158(4):842–844.
vonBoehmerH,BuerJ,HansenW:Neuropilin-1:asurfacemarkerof 39. TakamatsuH,OkunoT,KumanogohA:Regulationofimmunecell
regulatoryTcells.EurJImmunol2004,34(3):623–630. responsesbysemaphorinsandtheirreceptors.CellMolImmunol2010,
21. KawamuraH,LiX,GoishiK,vanMeeterenLA,JakobssonL,Cebe-SuarezS, 7(2):83–88.
ShimizuA,EdholmD,Ballmer-HoferK,KjellenL,etal:Neuropilin-1in 40. SarrisM,AndersenKG,RandowF,MayrL,BetzAG:Neuropilin-1expression
regulationofVEGF-inducedactivationofp38MAPKandendothelialcell onregulatoryTcellsenhancestheirinteractionswithdendriticcells
organization.Blood2008,112(9):3638–3649. duringantigenrecognition.Immunity2008,28(3):402–413.
22. NakamuraF,KalbRG,StrittmatterSM:Molecularbasisofsemaphorin- 41. BoissierMC,AssierE,BitonJ,DenysA,FalgaroneG,BessisN:RegulatoryT
mediatedaxonguidance.JNeurobiol2000,44(2):219–229. cells(Treg)inrheumatoidarthritis.JointBoneSpine2009,76(1):10–14.
23. VadaszZ,AttiasD,KesselA,ToubiE:Neuropilinsandsemaphorins-from 42. NeufeldG,KesslerO:Thesemaphorins:versatileregulatorsoftumour
angiogenesistoautoimmunity.AutoimmunRev2010,9(12):825–829. progressionandtumourangiogenesis.NatRevCancer2008,8(8):632–645.
24. KurosakaD,HiraiK,NishiokaM,MiyamotoY,YoshidaK,NodaK,UkichiT, 43. FukamachiS,BitoT,ShiraishiN,KobayashiM,KabashimaK,NakamuraM,
YanagimachiM,FuruyaK,TakahashiE,etal:Clinicalsignificanceofserum TokuraY:Modulationofsemaphorin3Aexpressionbycalcium
levelsofvascularendothelialgrowthfactor,angiopoietin-1,and concentrationandhistamineinhumankeratinocytesandfibroblasts.J
angiopoietin-2inpatientswithrheumatoidarthritis.JRheumatol2010, DermatolSci2011,61(2):118–123.
37(6):1121–1128. 44. AdlesicM,VerdrenghM,BokarewaM,DahlbergL,FosterSJ,TarkowskiA:
25. ClavelG,BessisN,LemeiterD,FardelloneP,MejjadO,MenardJF,PouplinS, Histamineinrheumatoidarthritis.ScandJImmunol2007,65(6):530–537.
BoumierP,VittecoqO,LeLoetX,etal:Angiogenesismarkers(VEGF, 45. TanakaS,SohenS,FukudaK:Aroleforhistaminereceptorsinrheumatoid
solublereceptorofVEGFandangiopoietin-1)inveryearlyarthritisand arthritis.SeminArthritisRheum1997,26(6):824–833.
theirassociationwithinflammationandjointdestruction.ClinImmunol 46. HashimotoA,TarnerIH,BohleRM,GaumannA,ManettiM,DistlerO,
2007,124(2):158–164. SteinmeyerJ,UlfgrenAK,SchulzA,GayS,etal:Analysisofvasculargene
26. KigelB,VarshavskyA,KesslerO,NeufeldG:Successfulinhibitionoftumor expressioninarthriticsynoviumbylaser-mediatedmicrodissection.
developmentbyspecificclass-3semaphorinsisassociatedwith ArthritisRheum2007,56(4):1094–1105.
