Resea ch A icle
Associa ion o Immunological Cell P o iles wi h Speci ic Clinical
Pheno ypes o Scle ode ma Disease
José Manuel López-Cacho,1Soledad Galla do,1Manuel Posada,2
Mi iam Ague i,1Da id Calzada,1Teodo o Mayayo,3Ma ía Luisa González-Rod íguez,4
An onio Ma ía Rabasco,4Ca los Lahoz,1,5 and Blanca Cá daba1,5
1Depa men o Immunology, IIS-Jim´
enez D´
ıaz Founda ion, Reyes Ca ´
olicos A enue 2, 28040 Mad id, Spain
2Ins i u e o Ra e Diseases Resea ch, Ca los III Ins i u e o Heal h, Eu oBioBank, and CIBERER, 28029 Mad id, Spain
3Sani-Red S.L, Ba celona Scien i ic Pa k, 08013 Ba celona, Spain
4Depa men o Pha maceu ical Technology, Facul y o Pha macy, Uni e si y o Se ille, 41012 Se ille, Spain
5CIBER o Respi a o y Diseases (CIBERES), 28029 Mad id, Spain
Co espondence should be add essed o Blanca C´
a daba; bca daba@ jd.es
Recei ed 7 Janua y 2014; Re ised 17 Feb ua y 2014; Accep ed 16 Ma ch 2014; Published 10 Ap il 2014
Academic Edi o : Richa d Gome
Copy igh © 2014 Jos´
eManuelL
´
opez-Cacho e al. This is an open access a icle dis ibu ed unde he C ea i e Commons
A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal wo k is
p ope ly ci ed.
This s udy aimed o sea ch he co ela ion among immunological p o iles and clinical pheno ypes o scle ode ma in well-
cha ac e ized g oups o scle ode ma pa ien s, compa ing o y-nine scle ode ma pa ien s s a i ied acco ding o speci ic clinical
pheno ypes wi h o y-nine heal hy con ols. Fi e immunological cell subpopula ions (B, CD4+and CD8+T-cells, NK, and
monocy es) and hei espec i e s ages o apop osis and ac i a ion we e analyzed by low cy ome y, in samples o pe iphe al blood
mononuclea cells (PBMCs). Analyses o esul s we e s a i ied acco ding o disease s age, ime since he diagnosis, and isce al
damage (pulmona y ib osis, pulmona y hype ension, and ca diac a lic ion) and by ime o ea men wi h co icos e oids. An
inc ease in he pe cen ages o monocy es and a dec ease in he B cells we e mainly ela ed o he disease p og ession. A gene al
apop osis dec ease was ound in all pheno ypes s udied, excep in localized scle ode ma. An inc ease o B and NK cells ac i a ion
was ound in pa ien s diagnosed mo e han 10 yea s ago. Speci ic cell popula ions like monocy es, NK, and B cells we e associa ed
wi h he ype o a ec ed o gan. This s udy shows how, in a he e ogeneous disease, p ope pa ien ’s s a i ica ion acco ding o clinical
pheno ypes allows inding speci ic cellula p o iles. Ou da a may lead o imp o emen s in he knowledge o p ognosis ac o s and
o aid in he analysis o u u e speci ic he apies.
1. In oduc ion
Scle ode ma is a a e au oimmune disease o unknown
e iology which a ec s housands o people a ound he wo ld.
I sp e alenceises ima ed obebe ween15and35cases
pe 100000 inhabi an s [1–3]. I s i s symp oms can be seen
a ound he hi d and ou h decade o he li e bu , in some
cases, symp oms can exis o se e al yea s wi hou a co ec
diagnosis. Scle ode ma is h ee imes mo e common in
women hanmen.Thediseaseisno linkedinanyconsis en
way o ace, season, geog aphy, occupa ion, o socioeconomic
s a us. En i onmen al e iologies a e none heless possible [1].
Scle ode ma is a complex au oimmune disease cha ac e -
ized by ib osis in all he o gans, al hough i s name is de i ed
om he ib osiso skincausedby hedisease.Damagein he
endo helium seems o be he ini ial lesion esponsible o he
cascade o e en s ha esul s in he disease [4,5]leading h ee
main ypes o al e a ions: ascula occlusion, immune sys em
al e a ions, and connec i e issue p oli e a ion.
Fib osis o he in e nal o gans leads o espi a o y p ob-
lems, dysphagia, bowel al e a ions, and kidney and ca diac
dys unc ions; hese complica ions lead o ma ked disabili y,
loss o quali y o li e, and high a es o mo ali y [1]. The e
a e wo main ypes o scle ode ma, localized and sys emic.
Hindawi Publishing Co po a ion
BioMed Resea ch In e na ional
Volume 2014, A icle ID 148293, 8 pages
h p://dx.doi.o g/10.1155/2014/148293
2BioMed Resea ch In e na ional
Localized scle ode ma a ec s mainly he skin, while sys emic
scle ode ma may a ec many pa s o he body. Localized
scle ode ma (LSc) can be classi ied as mo phea, linea , o
linea “en coup de sab e.” Sys emic scle ode ma (SSc) can
be di ided in o h ee majo sub ypes, limi ed cu aneous sys-
emic scle osis (lcSSc), di use cu aneous sys emic scle osis
(dcSSc) (scle osis o p oximal ex emi ies, unk, and ace),
and sys emic scle osis sine scle ode ma (o gan ib osis only;
no skin hickening).
Di e ences ha e been desc ibed in immunological cell
subpopula ions in pa ien s wi h di e en ypes o com-
plica ions and isce al in ol emen [6–8]. The esul s o
s udies in scle ode ma pa ien s which ha e in es iga ed
he numbe and he pe cen ages o lymphocy es [9–12],
hei ac i a ion [13,14], and apop osis s a es [14,15]ha e
shown some disc epancies. Reasons o hese disc epancies
a e no clea ly appa en bu could be ela ed o di e ences
in sub ype, s age, and ac i i y o he disease, demog aphic
cha ac e is ics o he pa ien s, and me hodological issues
in ob aining and analyzing pa ien samples [15]. To cla i y
hese disc epancies, we ha e s udied he ela ionship be ween
clinical scle ode ma pheno ypes and he di e en cellula
subpopula ions in well-cha ac e ized g oups o scle ode ma
pa ien s, compa ing hei esul s wi h heal hy con ols and
s a i ying hem acco ding o scle ode ma sub ypes, ime
since he diagnosis o he disease, and p esence o compli-
ca ions (pulmona y ib osis, pulmona y hype ension, and
ca diac a lic ion) and by ime wi h co icos e oids ea men .
Clinical cha ac e is ics like gas oesophageal e lux, digi al
ulce s, join in ol emen , scle ode ma enal c isis, o Rodnan
o al skin [16] sco e we e no conside ed pheno ypic s a a in
his s udy.
2. Ma e ials and Me hods
2.1. Subjec s. Scle ode ma pa ien s and con ols we e asce -
ained om Spanish Associa ion o Scle ode ma (AEE), wi h
he collabo a ion o he Ins i u e o Ra e Diseases Resea ch
(IIER), Ins i u o de Salud Ca los III (ISCIII), and he Spanish
Fede a ion o Ra e Diseases (FEDER). As all scle ode ma
pa ien s we e p e alen cases, we alida ed hei diagnosis
checking ha hey me he classi ica ion c i e ia o SSc
es ablished by he ARA in 1980 [17] ins ead o he new one
es ablished in 2013 by he ACR/EULAR [18].
The iden i ica ion o he di e en ypes o scle ode ma
as well as he o gan damages was based on clinical ea u es
ex ac ed om he clinical eco ds which we e made by
heuma ologis s and/o in e nal medicines specialis s a hos-
pi als o Spain. Time elapsed since he onse o he i s symp-
oms o he diagnosis o he disease was es ima ed h ough
elephone su eys, wi h he cases who accep ed o pa icipa e
in his s udy. Clinical medical eco ds we e e ised o con i m
some da es, when necessa y c i e ia o pulmona y ib osis,
hea lesions, and pulmona y hype ension we e ob ained
om echoca diog aphy and/o pulmona y unc ion es s
s a ed in clinical eco ds.
Con ols we e selec ed acco ding o he age and gende
o each pa ien ec ui ed who was esponsible o p o iding
Table 1: Pa icipan dis ibu ion by subse s.
Compa ison G oup Pa ien s
Con ols CONT 49
Pa ien s PAT 49
Type o scle ode ma
LSc 6
dcSSc 32
lcSSc 11
Time since he diagnosis
o he disease (yea s)
0–3 13
4–10 15
>11 21
T ea men wi h
co icoids (yea s)
No 21
0–3 17
>311
Pulmona y ib osis No 26
Yes 23
Ca diac a lic ion No 42
Yes 7
Pulmona y hype ension No 31
Yes 18
his/he own con ols among his/he iends and/o neighbo -
hoods. Con ols demons a ed no clinical indings sugges i e
o in ec ions, alle gic mani es a ions, o immunological dis-
o de s.
Fo y-nine scle ode ma pa ien s (43 emales and 6 males)
and 49 heal hy subjec s (26 emales and 23 males) we e
included in he s udy. Al hough he e was a highe p esence o
emales in scle ode ma pa ien s han in con ol g oup (87.7%
e sus 53.06%), he analysis acco ding o sex did no show any
s a is ically signi ican associa ion wi h cellula popula ion
s udied. The mean age ±s anda d de ia ion in he o al
g oup o pa ien s was 48.0 ± 10.9 yea s and 48.4 ± 13.1 yea s
o con ols (Table 1). Du ing he assays, hi een pa ien s
we e ecei ing co icos e oid ea men (11 wi h p ednisone,
1 wi h me hylp ednisolone, and 1 wi h de lazaco ), 4 pa ien s
we e unde going ea men wi h o he immunosupp essi e
d ugs, and 11 pa ien s we e ea ed wi h a combina ion o
an immunosupp essi e wi h a co icos e oid. None o he
pa ien s ook he medica ion in he 12 hou s be o e he
samples we e collec ed. All pa icipan s signed an in o med
consen in which hey ag eed o pa icipa e in he p ojec . The
p ojec was also app o ed by he E hics Commi ee o bo h
pa icipa ing ins i u ions (Ins i u o de Salud Ca los III and
IIS-Fundaci´
on Jim´
enez D´
ıaz).
2.2. Isola ion o Pe iphe al Blood Mononuclea Cells (PBMCs).
PBMCs om dono s we e isola ed om enous blood by
densi y g adien sedimen a ion on Lymphop ep (Come cial
Ra e , Za agoza, Spain) ollowing he manu ac u e ’s ins uc-
ions and esuspended in eezing medium con aining 90%
FBS(Lonza,Ve ie s,Belgium)and10%DMSO(Sigma-
Ald ich, S . Louis, USA). The cells we e aliquo ed in o
c yogenic ialsands o edinliquidni ogenun iluse.
PBMCswe e hawedinRPMI1640supplemen edwi h
BioMed Resea ch In e na ional 3
FL4 log: FL4 log loga i hmic
FL3 log: FL3 log loga i hmic
FL4 log: FL4 log loga i hmic
FL4 log: FL4 log loga i hmic
FL1 log: FL1 log loga i hmic
FL3 log: FL3 log loga i hmic
FL2 log: FL2 log loga i hmic
FL5 log: FL5 log loga i hmic
FL3 log: FL3 log loga i hmic
11E1 1E2 1E3 1E4
1
1E1
1E2
1E3
1E4
11E1 1E2 1E3 1E4
1
1E1
1E2
1E3
1E4
ANEX-V+CD25+
11E1 1E2 1E3
SS lin: SS lin Exp-SSC low
0
100
200
300
500
11E1 1E2 1E3
1
1E1
1E2
11E1 1E2 1E3
1
1E1
1E2
1E3
CD3+CD8+
CD3+CD4+CD8+
Monocy es
CD4+SSC
B cells (CD19+)
NK cells (CD56+)
CD3+CD4+
CD3+cells
Figu e 1: Cha ac e iza ion o cellula subpopula ions analyzed by low cy ome y. FL1: Annexin-V+cells; FL2: CD3+o /and CD56+cells;
FL3: CD4+cells; FL4: CD19+o CD8+cells; FL5: CD25+cells; SSC: side-sca e de ec o .
5% FBS inac i a ed, 1% L-glu amine, 1% peni-s ep omycin,
and 1 mM sodium py u a e (Flow Labo a o ies, I ine, UK)
and a e wo washed, he PBMCs we e esuspended in
saline bu e and s ained acco ding ecommenda ions o he
speci ic comme cial an ibody. Quan i ica ion and iabili y
we e de e mined by ypan blue exclusion. Be o e doing
apop osis and ac i a ion analysis, con ols wi h eeze PBMCs
samples we e ca ied ou o es ing quali y o assays. Samples
included o analysis had a iabili y o a leas 90%. All
biological samples we e collec ed om he Ins i u e o Ra e
Diseases Resea ch Biobank, belonging o Eu opean Ra e
Diseases Biobank-Eu oBioBank.
2.3. Immunopheno yping. Cellula pheno ype was analyzed
by low cy ome y using a single PBMCs sample labeled
wi h a i e-colo combina ion o monoclonal an ibodies o
Annexin-V-FITC (FL-1) (Re : ANXVKF-100T, Inmunos ep,
Salamanca, Spain), CD3-PE (Re : CYT-3PE5) and CD56-PE
conjuga ed (FL-2) (Re : CYT-56PE) (Vi o-Cy ognos, Sala-
manca, Spain), CD4-ECD (FL-3) (Re : 6604727, Beckman
Coul e , Izasa, Ba celona, Spain), CD8-PC5 (Re : CYT-8C2)
and CD19-PC5 conjuga ed (FL-4) (Re : CYT-19C2) (Vi o-
Cy ognos, Salamanca, Spain), and CD25-PC7 (FL-5) conju-
ga ed (Re : PN A52882, Beckman Coul e , Izasa, Ba celona,
Spain) by using Cy omics FC500 and CXP so wa e (Beck-
man Coul e , B ea, USA). Unlike o he s udies, he di e -
en signal in ensi ies o his combina ion o luo och omes
allowed us o analyze se e al clus e s o di e en ia ion a he
same ime using only one de e mina ion (Figu e 1). A leas
5,000 e en s we e acqui ed in a lis mode and all he da a we e
analyzed by cell ques so wa e (CXP Beckman Coul e ).
Fi e-colo low cy ome y analysis was pe o med as
ollows. B ie ly, 2⋅105PBMCs we e labeled wi h 20 𝜇L o each
monoclonal an ibody (CD3-PE, CD56-PE, CD4-ECD, CD8-
PC5, CD19-PC5, and CD25-PC7 conjuga ed) and placed in
an ice-wa e ba h o 20 minu es.
A e washingwi hPBS,20𝜇L o Annexin-V-FITC was
deli e ed and he ubes we e placed in an ice-wa e ba h o
15 minu es in da kness. Then, he cells we e esuspended in
200 𝜇L o binding bu e (PBS) o immedia e low cy ome y
analysis.
Li e PBMCs we e elec onically ga ed by o wa d-
and igh -angle sca e s; di e en ypes o cells we e hen
ga ed and he pe cen age and in ensi y o exp ession we e
e alua ed. When he ga ed cells we e unce ain o e y
ew, he da a we e excluded. Nega i e con ols included
a human iso ype-ma ched non ele an immunoglobulin:
Mouse IgG2a-PE (CYT-IC006PE, Cy ognos) and IgG2b-
PE (CYT-IC007PE, Cy ognos), IgG1-PECy5 Mouse Iso ypic
Con ol (CYT-IC005C, Cy ognos), Mouse IgG2a- PC7 (PN
4BioMed Resea ch In e na ional
CONT PAT LSc lcSSc dcSSc NO PF PF NO PH PH
10
20
30
40
B lymphocy es
Cells (%)
∗
∗
∗
∗
‡
Figu e 2: Box-plo o B lymphocy es. ∗S a is ically signi ican ela-
i e o con ols. ‡S a is ically signi ican ela i e o pa ien s wi hou
pulmona y hype ension.
A12692, Beckman Coul e ), and Mouse IgG1-ECD Iso ypic
Con ol (A07797, Beckman Coul e ). Figu e 1 (uppe panel)
summa izes he selec ion o cell popula ions s udied: pe -
cen ages o T lymphocy es CD3+CD4+and CD3+CD8+we e
e alua ed o he o alnumbe o CD3
+cells. Pe cen ages o B
cells (CD3−CD19+), NK cells (CD3−CD56+), and monocy es
we e e alua ed ega ding o al numbe o cells. Monocy es
we e ga ed based on CD3−CD4+exp ession and high side-
sca e (SSC).
A e he de e mina ion o cellula subpopula ions,
he pe cen ages o apop osis (Annexin-V+) and ac i a ion
(CD25+exp ession) o each subpopula ion we e e alua ed
ega ding hei espec i e o e all numbe o cells (lowe panel
o Figu e 1).
2.4. S a is ical Analysis. Global analysis was pe o med by
K uskal-Wallis es and compa isons be ween g oups we e
pe o med using a nonpa ame ic wo- ailed Mann-Whi ney
es o independen a iables. A 𝑃 alue o <0.05 was used.
Weused heRp og am o hes a is icalanalysisand hebox-
plo s design.
3. Resul s
Table 2 shows hemedianso hepe cen ageso each ypeo
cellula subpopula ions s udied, as well as hei ac i a ion o
apop osis s ages, g ouped by clinical pheno ype.
Signi ican di e ences we e de ec ed in scle ode ma
pa ien s when we compa e hei esul s wi h he con ol
g oup. We ound a dec ease in he pe cen age o B lympho-
cy es and highe alues o monocy es. We did no ind any
di e ence in ac i a ion s a es, al hough o apop osis alues
we obse ed a dec ease in all he subse s o cells compa ed o
con ols.
Acco ding o scle ode ma pheno ypes, we ound a p o-
g essi e educ ion in he pe cen ages o B lymphocy es in
he mos se e e pheno ypes o he disease (Figu e 2); his
dec ease in he di use sys emic a ian (𝑃 < 0.01)compa ed
CONT PAT LSc lcSSc dcSSc NO PF PF NO PH PH
20
40
60
80
O e all apop osis
Apop osis (%)
∗
∗
∗∗∗
∗
∗
†
Figu e 3: Box-plo o o e all apop osis ega ding scle ode ma
ype and p esence o PH and PF. ∗S a is ically signi ican ela i e
o con ols. †S a is ically signi ican ela i e o pa ien s wi hou
pulmona y hype ension.
CONT PAT NO PF PF NO PH PH
0
10
20
30
40
50
60
Ac i a ed NK cells
Cells (%)
∗
∗
Figu e 4: Box-plo o ac i a ed NK cells. ∗S a is ically signi ican
ela i e o con ols.
o con ols was s a is ically signi ican . A s a is ically signi -
ican inc ease in monocy es was shown ela i e o con ols
(3.35%) only in pa ien s a ec ed by sys emic scle ode mas
(7.41% in limi ed o m and 7.91% in di use o m). No
di e ence in cell ac i a ion was ound among scle ode ma
pheno ypes. As o o e all apop osis, we ound a dec ease in
sys emic bu no in localized scle ode mas (Figu e 3). Apop-
osis aluesbycellula popula ion(Table 2) we e dec eased
in se e al cellula subse s in sys emic scle ode mas. The
educ ion was in all he subse s analyzed in he di use o m
and B cells and in monocy es in he limi ed o m. Localized
scle ode mas showed an apop o ic dec ease in monocy es.
In pa ien s wi h pulmona y ib osis (PF), we ound a
educ ion in he pe cen ages o B lymphocy es (Figu e 2)and
an inc ease o ac i a ed NK cells (Figu e 4). Pa ien s wi h
and wi hou ib osis showed inc eased monocy e pe cen ages
(Table 2) and simila apop o ic p o iles. We ob ained educed
pe cen ages in all cell subpopula ions excep in CD4 T-cells
in pa ien s wi h PF.
BioMed Resea ch In e na ional 5
Table 2: Cell subpopula ion pe cen ages (medians) s a i ied by subse s.
Cell
popula ion
O e all Type Pulmona y
ib osis
Pulmona y
hype ension
Ca diac
a lic ion
Time since he
diagnosis o he disease
(yea s)
Co icos e oids
ea men (yea s)
Con ols
𝑛=49 Pa ien s
𝑛=49 LSc
𝑛=6 lcSSc
𝑛=11 dcSSc
𝑛=32 No
𝑛=26 Yes
𝑛=23 No
𝑛=31 Yes
𝑛=18 No
𝑛=42 Yes
𝑛=7 0–3
𝑛=13 4–10
𝑛=15 >10
𝑛=21 No
𝑛=21 0 o3
𝑛=17 >3
𝑛=11
Gene al
CD4+T-cells 65.04 62.46 62.36 66.17 63.84 62.36 63.84 65.66 62.19 62.46 68.35 64.16 63.84 61.91 62.31 62.46 65.46
CD8+T-cells 29.86 27.16 28.45 25.77 25.14 27.66 25.65 26.15 27.5 27.66 25.14 28.81 24.44 29.13 25.26 29.23 25.14
Bcells 15.62 11.14 16.19 12.13 10.56 14.02 9.97 16.87 5.98‡12.39 6.19 12.51 12.13 11.01 18.50 10.56 9.97
NK cells 11.09 11.73 10.09 9.74 11.77 9.89 12.4 10.51 12.7 11.65 12.34 9.43 11.65 12.34 14.81 9.89 10.39
Monocy es 3.35 7.41 4.65 7. 4 1 7. 9 1 7. 4 1 7. 2 5 7. 6 6 3.78 7. 2 5 1 2 . 81 6.87 7. 5 3 4.43 6.87 7.53 4.43
Ac i a ion
O e all 7.32 8.07 9.37 8.68 6.46 6.46 8.63 6.87 8.67 8.20 6.91 8.38 5.14 9.52∗8.67 8.68 6.46
CD4+T-cells 5.05 6.88 7.81 8.47 5.78 5.78 7.21 5.54 7.08 6.69 7.99 8.17 4.67 7.44 5.23 7.69 6.69
CD8+T-cells 4.34 5.66 7.00 5.29 4.49 5.29 8.25 5.08 8.28 6.09 4.17 6.61 3.87 7.09 7.56 6.90 4.17
B cells 9.79 11.61 13.11 5.41 11.98 11.87 11.48 11.33 15.69†11.87 10.00 12.19 9.03 13.48 8.54 13.48 14.71
NK cells 14.93 19.57 24.57 22.41 15.68 18.41 21.39 18.99 22.44 19.57 21.30 23.27 11.76 23.48 17.05 23.09 17.71
Monocy es 70.80 70.10 81.75 66.67 61.90 77.36 59.42 74.88 61.52 74.75 58.60 62.93 84.68 61.90 84.45 70.66 50.00
Apop osis
O e all 29.00 19.16 31.23 18.51 15.41 19.67 19.16 14.67 25.32†19.16 19.61 16.77 15.41 25.22 20.21 19.61 12.88
CD4+T-cells 22.56 17.48 22.61 13.97 11.05 17.66 17.48 11.10 21.88†17.48 21.36 13.23 11.05 22.79 18.44 18.22 11.05
CD8+T-cells 21.42 16.75 28.57 21.05 10.63 16.67 21.05 10.49 24.8 16.75 22.00 21.07 10.35 27.08 16.71 21.69 9.84
B cells 30.97 19.42 38.59 15.52 18.92 19.12 20.92 15.26 28.13†20.92 18.92 18.85 19.12 22.35 20.89 24.68 18.92
NK cells 45.68 35.43 68.02 37.93 30.77 37.93 32.67 33.10 45.32 36.69 32.67 37.31 27.99 40.81 31.18 47.54 33.33
Monocy es 80.00 62.90 59.67 65.97 62.90 58.58 66.67 58.32 67.48 62.90 62.04 57.44 62.04 66.26 62.93 66.41 55.88
Bold le e s indica e s a is ically signi ican esul s ela i e o con ols. ‡S a is ically signi ican compa ed o con ols and wi hou pulmona y hype ension. †S a is ically signi ican ela i e o pa ien s wi hou
pulmona y hype ension. ∗Indica e hose da a s a is ically signi ican compa ed o ou and en yea s om he diagnosis o he disease.

6BioMed Resea ch In e na ional
In he g oup o pa ien s wi h pulmona y hype ension
(PH), we ound he lowes pe cen ages o B cells (Figu e 2).
This g oup was he only one wi hou an inc ease o monocy es
(Table 2). We also ound signi ican inc eases o ac i a ed B,
NK, and CD8+cells. The inc ease o CD8+cellsisal e ed
like in pulmona y ib osis. Rega ding apop osis, we ound
dec eases in all he subpopula ions o pa ien s wi hou pul-
mona y hype ension when compa ing hese subpopula ions
o con ols. In addi ion, we ound s a is ically signi ican
di e ences when compa ing absence and p esence o PH,
wi hlowe apop osis oundin heabsenceo pulmona y
hype ension (Table 2).
We ound ew signi ican di e ences in pa ien s a ec ed
by ca diac a lic ion (CA) p obably due o he low numbe
o pa ien s es ed (𝑛=7);weonly oundaninc easeo
monocy es (Table 2).
Rega ding ime since he diagnosis o he disease, we
only ound an inc ease o monocy es in pa ien s diagnosed
om 4 o 10 yea s (Table 2). Pa ien s diagnosed mo e han
10 yea s p e iously showed an inc ease in he pe cen ages
o ac i a edBandNKcells; hese esul swe e e ysimila
o hose ob ained in pa ien s diagnosed om 0 o 3 yea s
(Table 2). We also ound he lowes pe cen ages o apop osis
in he g oup o pa ien s diagnosed om 4 o 10 yea s ago
(Table 2), bu a p og essi e eco e y is p oduced wi h ime,
excep o B cells and monocy es.
Rega ding o he ea men ime wi h co icos e oids,
he ea men dec eases he pe cen ages o B cells and, in
pa ien s ea ed du ing mo e han 3 yea s, a educ ion o
he pe cen ages o monocy es and hei ac i a ion s a e a e
p oduced (Table 2). In his g oup we also ob ained he
smalles pe cen ages o apop osis (Table 2). In addi ion, he
g oup o pa ien s diagnosed om ze o o h ee yea s had
highe pe cen ages o ac i a ed NK cells.
4. Discussion
The e ha e been con lic ing esul s conce ning changes in he
immune cell subpopula ions in ol ed in scle ode ma [11,19–
21] due o he he e ogenei y o he disease and di e ences in
s udy me hods. In o de o shed ligh on his issue, we ha e
s udied a well-de ined clinical popula ion, analyzing bo h
he cell subpopula ions and hei ac i a ion and apop osis
s ages and also co ela ing he da a wi h di e en clinical
pheno ypes.
We ob ained no mal alues o T lymphocy es, as has been
desc ibed by o he au ho s [11,20–22]. Howe e , we ound
a educ ion o B-cells in he wo se-p ognosis si ua ions o
hediseasebu highe pe cen ageso monocy esinalmos
all SSc subse s. These esul s can be also co ela ed wi h he
condi ions o isce al in ol emen ha igge s he wo s
p ognosis (Figu e 2 and Table 2).
The monocy e ac ion o pe iphe al blood con ains
p ecu so s o po en ial egula o s o ib osis [23]suchas
ib ocy es and mac ophages [18,24,25]. Se e al s udies
ob ained unc ional abno mali ies and inc eased numbe s o
hese cells in scle ode ma pa ien s [23,26–29]. Ou esul s
a e consis en wi h many da a ha indica e he associa ion
be ween ele a ed le els o pe iphe al blood ib ocy es and
monocy es and di e se o ms o o gan emodeling such as
enal ib osis, ci hosis, o di e en ypes o pulmona y ib o-
sis [29]. An abno mal emodeling in scle ode ma is usually
mo e common in sys emic scle ode mas [30]. Howe e , ou
monocy es esul s mus be conside ed wi h cau ion because
monocy es we e no de ec ed di ec ly h ough a CD14 ma ke
and, ins ead, we e selec ed indi ec ly, as CD3−CD4+,and
some monocy es canno exp ess CD4.
The ac ion o B cells in scle ode ma could be ela ed o
hei unc ions in he egula ion o he immune esponse
[31] and an ibody p oduc ion and abno mali ies in hem can
induce o de elop au oimmune diseases like scle ode ma.
Analyzing he ac i a ion s a es, we ob ained a highe
o e all ac i a ion in pa ien s in whom he ime elapsed since
diagnosiswasg ea e o inpa ien swi h isce alin ol emen
(especially in B and NK cells). Some au ho s de e mine ha
su i al a es o scle ode ma pa ien s begin hei decline in
hepe iodo imeo ou o enyea ssince heygo o he
hospi al [32,33]. As de ec ed in ou s udy, he de elopmen
o se ious isce al in ol emen po ends ea ly mo ali y in
pa ien s wi h scle ode ma [32,34,35] and is concomi an wi h
ele a ed ac i a ion s a es o immune cells [28,36,37]. The ole
played by NK cells in au oimmune diseases is bo h a disease-
con olling and a disease-p omo ing ole and NK cells may
be implica ed in he onse , main enance, o p og ession o
hese diseases [38], hus sugges ing hei in ol emen in he
scle ode ma [39].
Ac i a ion ma ke s like CD25 play a ole in cell con ac
in e ac ions be ween T-cells and o he cells, including ib ob-
las s. Howe e , he analysis o o he lymphocy es ac i a ion
ma ke sasCD69o MHCClassIIcouldbein e es ing,
especially o de e mining monocy e ac i a ion. Inclusion o
cy okine p oduc ion om hese cell popula ions could also
enhance in o ma ion abou hei s a es o ac i a ion.
Apop osis alues may be di icul o e alua e due o he
me hod o conse ing he cells. In his s udy, as ozen
cells we e used, he eezing e ec was checked and was
homogenous in all samples. Annexin V s aining p ecedes
he loss o memb ane in eg i y which accompanies he la e
s ages o cell dea h esul ing om ei he apop o ic o nec o ic
p ocesses, o ha was selec ed as cellula dea h ma ke .
We ound a dec ease in di e en subse s in he disease,
possibly because o apop osis esis ance in he cells o scle o-
de ma pa ien s. Apop osis esis ance is he p ima y mecha-
nism leading o he de elopmen o ib o ic lesions and could
be one o he easons why some ypes o scle ode mas ha e
mo e isce al ib osis and wo se p ognosis, especially in he
case o di use o ms, whe e he apop osis a es a e he lowes .
Some au ho s [40] ha e desc ibed ha , du ing la e phases o
sys emic scle osis, in lamma o y e en s become less in ense
and he immune esponse ac s as a low-g ade ampli ie o
ib ogenesis and mic oangiopa hy, p esen ing a signi ican
he apeu ic challenge. This could explain he di e ences ha
we ha e ound be ween he pa ien s analyzed acco ding o
imesincediagnosis:adiminu ionin hebeginningo he
disease, which could coincide wi h he endo helial damage;
a e ou o en yea s, he disease could coincide wi h he
de elopmen o ib osis in he in e nal o gans, whe e we ha e
BioMed Resea ch In e na ional 7
ob ained apop osis diminu ions; and, inally, un il abou en
yea s ollowing diagnosis, he apop osis ends o inc ease and
he ib osis ends oslowdown.
In pa ien s diagnosed mo e han en yea s p e iously,
he educ ion in he apop osis o B cells and monocy es is
pa allel o an inc ease in he o e all ac i a ion, especially in
he ac i a ion o B and NK cells. This coincides wi h dec eases
in he su i al a es o he scle ode ma pa ien s [32,33]and
o gan a lic ion ends o appea in he la e-onse s ages o he
disease [30]. The de ec ed al e a ions in hese subpopula ions
couldha esomein luenceonsu i al a esandcouldbe
use ul in he p ognosis o he disease.
The usual ea men o scle ode ma—especially o o gan
ib osis—[30] is based on immunosupp essi e d ugs and co -
icos e oids ha inc ease he apop osis o he in lamma o y
cells [41]. In ou esul s, he ea men seems o inc ease
heapop osisin heg oup ea edwi hco icos e oids om
ze o o h ee yea s bu , in pa ien s wi h mo e han h ee
yea s ea ed, he si ua ion e e s o diminished pe cen ages.
This could be one o he easons ha make ea men wi h
co icos e oids ine icien in pa ien s who ha e had he
disease o many yea s. In addi ion o his, wo hings mus
be aken in o accoun : bo h kinds o d ugs can in e e e wi h
he ac ion o B cells, al hough hey usually do no educe
hei numbe s and co icos e oids p oduce an inhibi o y
e ec in NK cells [42] bu hese cells ha e a lowe sensi i i y
o hese kinds o d ugs [43]. These da a coincide wi h ou
esul s whe e mo e ime is needed o educe hei ac i a ion.
Howe e , co icos e oids a e use ul in monocy es, p oducing
a educ ion in he pa ien s wi h mo e ime wi h he disease.
5. Conclusions
In conclusion, an o e all dec ease in apop osis and in he
numbe s o B cells oge he wi h an inc ease o monocy es
and ac i a ed B and NK cells is di ec ly co ela ed wi h he
se e i y o he disease. These esul s sugges ha hese cells
con ibu e o he immunological abno mali ies obse ed in
scle ode maandcouldannounce heappea anceo compli-
ca ions o a wo sening in he disease.
Con lic o In e es s
The au ho s decla e ha hey ha e no inancial ela ionships
ela ed o any p oduc s in ol ed in his s udy ha migh lead
o a con lic o in e es s.
Acknowledgmen s
The au ho s would like o hank he Spanish Fede a ion
o Ra e Diseases (FEDER, Fede aci´
on Espa˜
nola de En e -
medades Ra as) and he Spanish Associa ion o Scle ode ma
(Asociaci´
on Espa˜
nola de Escle ode mia, AEE) o hei assis-
ance in ec ui ing olun ee s. They also hank he Spanish
Ra e Diseases Biobank, Eu oBioBank, pa ne o hei sup-
po in he biological samples managemen . They also would
like o acknowledge he help o he IIS-Fundaci´
on Jim´
enez
D´
ıaz and especially Ignacio Mahillo om he Epidemiology
Depa men o assis ance wi h s a is ical analysis. They also
wouldlike o hank heUni e si yo Se ille.Thiswo kwas
suppo ed by he en e p ise By Bio ech & Science by G an s
om Co po aci´
on Tecnol´
ogicaAndaluzaandAgenciaIDEA
(no. exp. 34323). Mi iam Ague i and Da id Calzada we e
suppo ed by Fundaci´
on Conchi a R´
abago, Mad id, Spain.
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Hindawi Publishing Co po a ion
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Pa kinson’s
Disease
E idence-Based
Complemen a y and
Al e na i e Medicine
Volume 2014
Hindawi Publishing Co po a ion
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