Differ ing phagocytic function of monocytes and neutr ophils in Chagas’
car diopathy accor ding to the pr esence or absence
of congestive hear t failur e
Diferenciada função de monócitos e neutrófilos na cardiopatia chagásica segundo
a presença ou ausência de insuficiência cardíaca congestiva
Mar ia Imaculada Muniz-Junqueir a
1, Lícia Mar ia Mota
1, Rodr igo Bar bosa Air es
1and Luiz Fer nando Junqueir a Júnior
2ABSTRACT
We e va lu a te d th e in vitr o p h a go c yti c f u n c ti o n a n d th e p ro d u c ti o n o f m i c ro b i c i d a l o x yge n ra d i c a ls b y m o n o c yte s a n d n e u tro p h i ls o f 9 Ch a ga s’ h e a rt d i se a se su b je c ts wi th h e a rt f a i lu re a n d 9 wi th o u t th e syn d ro m e i n c o m p a ri so n wi th 1 1 he a lthy su b je c ts, b y a sse ssi n g pha go c yto si s o f Sac c har o myc es c er evisiae a n d NBT re du c ti o n b y pe ri phe ra l b lo o d pha go c yte s. Ph a go c yti c i n d e x o f m o n o c yte s o f c h a ga si c s wi th o u t h e a rt f a i lu re wa s si gn i f i c a n tly 6 .7 a n d 1 0 .6 ti m e s lo we r th a n th o se o f c o n tro ls a n d c h a ga si c s wi th th e c o n ge sti ve syn d ro m e , re sp e c ti ve ly, d u e to a le sse r e n ga ge m e n t i n p h a go c yto si s a n d to a n i n a b i li ty o f th e se c e lls to i n ge st p a rti c le s. Ne u tro p h i ls a lso sh o w i n c h a ga si c s wi th o u t h e a rt f a i lu re PI 1 1 .2 a n d 1 9 .8 ti m e s lo we r th a n th a t o f c o n tro ls a n d c h a ga si c s wi th h e a rt f a i lu re , re sp e c ti ve ly. Th e p e rc e n t o f NBT re d u c ti o n wa s n o rm a l a n d si m i la r f o r th e th re e gro u p s. Ba la n c e d o p p o si te e f f e c ts o f c a rd i o va sc u la r a n d i m m u n e d i stu rb a n c e s m a y b e a c ti n g i n Ch a ga s’ d i se a se su b je c ts wi th h e a rt f a i lu re p a ra d o x i c a lly re c o ve ri n g th e a lte re d p h a go c yti c f u n c ti o n .
Ke y-words: Ch a ga s’ h e a rt d i se a se . Ca rd i a c f a i lu re . Ph a go c yto si s. Ni tro b lu e te tra zo li u m te st. Ne u tro p h i ls. Mo n o c yte s.
RESUMO
A fu n ç ã o fa go c itá ria e a pro du ç ã o de ra dic a is m ic ro b ic ida s de o xigê n io pe lo s m o n ó c ito s e n e u tró filo s fo ra m a va lia da s e m 9 pa c ie n te s c o m c a rdio pa tia c ha gá sic a c o m in su fic iê n c ia c a rdía c a c o n ge stiva e 9 se m a sín dro m e e m c o m pa ra ç ã o c o m 11 in divídu o s c o n tro le s n o rm a is pe lo te ste de fa go c ito se de Sac c haro myc es c erevisiae e re du ç ã o do nitroblue tetrazolium pe lo s f a gó c i to s d o sa n gu e p e ri f é ri c o . O í n d i c e f a go c i tá ri o d o s m o n ó c i to s d o s c h a gá si c o s se m i n su f i c i ê n c i a c a rd í a c a f o i sign ific a n te m e n te 6,7 e 10,6 ve ze s m e n o r do q u e o do s c o n tro le s e c ha gá sic o s c o m a sín dro m e c o n ge stiva , re spe c tiva m e n te , de vido a o m e n o r e n vo lvim e n to n a fa go c ito se e a m e n o r c a pa c ida de de sta s c é lu la s de in ge rire m pa rtíc u la s. No s c ha gá sic o s se m in su fic iê n c ia c a rdía c a o s n e u tró filo s ta m b é m a pre se n ta ra m o IF 11,2 e 19,8 ve ze s m e n o r q u e o s c o n tro le s e c ha gá sic o s c o m in su fic iê n c ia c a rdía c a , re spe c tiva m e n te . A po rc e n ta ge m de re du ç ã o do NBT fo i n o rm a l e sim ila r pa ra o s trê s gru po s. Efe ito s o po sto s e q u ilib ra do s da s disfu n ç õ e s de vida s à s a lte ra ç õ e s c a rdio va sc u la re s e im u n e s po de m te r a tu a do n o s pa c ie n te s c ha gá sic o s c o m in su fic iê n c ia c a rdía c a pa ra do xa lm e n te re c u pe ra n do a fu n ç ã o fa go c itá ria a lte ra da .
Pal avr as-chave s: Ca rd i o p a ti a c h a gá si c a . In su f i c i ê n c i a c a rd í a c a . Fa go c i to se . Te ste d o nitr o b lue te tr azo lium. Ne u tró f i lo s. Mo n ó c i to s.
1 . Lab o r ató r io de Imuno lo gia Ce lular da Fac uldade de Me dic ina da Unive r sidade de B r asília, B r asília, DF. 2 . Se r viç o de Car dio lo gia do Ho spital Unive r sitár io de B r asília da Unive r sidade de B r asília, B r asília, DF.
Addre ss to: Dra. Maria Imac ulada Muniz-Junqueira. Laboratório de Imunologia Celular, Fac uldade de Medic ina, Universidade de B rasília, 7 0 9 1 0 -9 0 0 B rasília, DF, B rasil. Fax: 5 5 6 1 2 7 3 -3 9 0 7
e - mail: mimj unq ue ir a@ unb . b r Re c e b ido par a pulic aç ão e m 2 8 /1 1 /2 0 0 3 Ac e ito e m 2 7 /9 /2 0 0 4
Chagas’ disease is a lifelong endemic infec tious c ondition
c aused by the intrac ellular hemoflagellate parasite Trypa no so m a
cruzi that affec ts mainly the heart and digestive trac t c ausing an ac ute loc al inflammatory reac tion whic h may be followed by a
long-lasting progressive c hronic c ellular and humoral immune
process resulting in variable lesion with functional impairment
mainly of the common and specialized myocardial cells, myoenteric fibers, peripheral intrinsic autonomic ganglia and nerve fibers,
and muscarinic and beta-adrenergic cell receptors2 1 2 2 2 6 4 3 4 4 4 6.
o f c o ntr o ve r sy, with so me e vide nc e sugge sting a lo ng-lasting
auto immune r e ac tio n tr igge r e d in the ac ute phase , whe r e as o the r autho r s have po inte d to a c o ntinuo us immune stimulus
mec hanism r equir ing par asite per sistenc e dur ing the c hr o nic
phase1 2 2 4 3 4 4 7.
Chagas’ disease in its c hronic stage is a major c hallenge for
public health in South and Central Americ a c ountries, where it affec ts about 1 6 to 1 8 million people under different c linic al
forms, denominated indeterminate, exc lusive c ardiac or digestive
and c ombined c ardiac plus digestive5 2. It presents high morbidity
and mortality princ ipally due to a pec uliar c hronic c ardiopathy that o c c urs in abo ut 3 0 -4 0 % o f infec ted individuals, whic h
c ommonly results in c ongestive heart failure of variable severity,
c onstituting the main c ause of disability and death2 0 4 2 4 4.
Congestive heart failure is a c omplex func tional disturbanc e
that implic ates a multiplic ity o f inter-playing hemo dynamic , autonomic nervous, metabolic , hormonal, hydro-elec trolytic and
immunologic al mec hanisms and fac tors among others1 7 1 9 3 0.
Cytokines, espec ially the pro-inflammatory ones, tumor nec rosis
fac tor-
α
, and IL-1 and IL-6 are some of the immunological factorsproduc ed by immune c ells7 1 6 involved in the syndrome of heart
failure1 2 2 7 3 0. Tumor nec rosis fac tor-
α
, a produc t of ac tivatedmac rophages, is inc reased in the serum of patients with severe
c ongestive heart failure4 8 5 1, and it has been suggested that this
c ytokine c an play an important role in the pathogenesis of this
syndrome2 2 9 3 2.
On the other hand, tumor nec rosis fac tor-
α
may dec reaseo r inc r e ase the phago c ytic c apac ity o f hum an m o no c yte s
de pe nding o n the c o nc e ntr atio n o f this c yto k ine3 9. Othe r
observations indic ate that c ytokines produc ed by monoc ytes and
neutrophils may also influence the heart mechanical function2 3 2.
In additio n, mo no c ytes and neutr o phils ar e the main c ells
involved in the defense against bac teria that may c ause infec tions, whic h might c omplic ate the c linic al c ourse of patients with heart
failure1 8. However, it is not yet known if the pathophysiologic al
c hanges o b ser ved in c o ngestive hear t failur e may alter the
func tion of c ells of the immune system.
Inc reased produc tion of tumor nec rosis fac tor-
α
in Chagas’disease patients with c hronic c ardiomyopathy1 0 and in c hronic
experimental Trypa no so m a cruzi infec tion3 3 has been observed.
Altered concentrations of other cytokines produced by monocytes
and neutrophils have also been observed in Chagas’ heart disease
subjec ts1 0 1 1. In addition, peripheral blood monoc ytes and heart
mac rophages play a c ruc ial role in the resistanc e to development
of Chagas’ disease2 5 3 1.
Therefore, it may be important to investigate the innate immune
function represented by the phagocytosis in Chagas’ heart disease
and its congestive heart failure syndrome to gain insights into the
overall pathophysiological mechanisms implicated, including that
concerned with conditions such as infectious pictures that may
occur in the course of this syndrome. Thus, we aimed to evaluate
the still unexplored phagocytic function and the production of
microbicidal oxygen radicals by monocytes and neutrophils in
Chagas’ heart disease subjects with and without congestive cardiac
failure in comparison with healthy control subjects.
MATERIAL AND METHODS
Study gr oups. The evaluation included 2 9 volunteer subjects distributed in three study groups: a) c ontrol group of healthy subjec ts ( n = 1 1 ) , 9 male and 2 female, aged 2 0 to 4 5 years
( median, 2 8 years) ; b) Chagas’ heart disease without c ongestive
c ardiac failure group ( n = 9 ) , 4 male and 5 female, aged 1 7 to
5 6 years ( median, 4 0 years) ; c ) Chagas’ heart disease with NYHA func tional c lass III-IV c ongestive c ardiac failure group ( n = 9 ) ,
8 male and 1 female, aged 1 6 to 5 8 years ( median, 4 8 years) .
The subjec ts of the groups did not differ statistic ally in their ages
( p > 0 .0 5 , Kruskal-Wallis test) . Possible influenc ing fac tors on the immunological function were ruled out in the control subjects
and none was using any drug. The c hagasic subjec ts with heart
disease inc luded had no other suspec ted or assoc iated disease.
Those with heart failure were taking c onventional drug therapy fo r the ir he ar t c o nditio n, suc h as, diur e tic s ( fur o se mide ) ,
vasodilators ( prazosin or hydralazine) , angiotensin c onverting
enzyme inhibitors ( captopril or enalapril) , aldosterone inhibitors
( spironolac tone) and antiarrhythmic s ( amiodarone) . Chagas’ h e a r t dis e a s e pa tie n ts h a d n o e pide m io lo gic a l, c lin ic a l,
elec troc ardiographic and ec hodopplerc adiographic evidenc e of
heart disease due to other etiology.
The diagnostic c riteria for exc lusive Chagas’ heart disease
we r e the pr e se nc e o f spe c ific po sitive se r o lo gic r e ac tio ns ( i n di r e c t i m m u n o fl u o r e s c e n c e ) a n d c a r di a c c l i n i c a l
manifestations with typic al alterations in the elec troc ardiogram
c harac teristic of the disease2 0 2 1 4 4. Congestive heart failure was
di a gn o s e d o n th e b a s i s o f th e c l i n i c a l a n d l a b o r a to r y manifestations of the syndrome. No c hagasic patient showed
dige stive sympto ms and all had e pide mio lo gic al histo r y o f
exposure to the transmission vec tor of the disease.
The ethic al rules of the Helsinki Dec laration and those from
the Brazilian National Counc il of Health for experimentation in human beings were stric tly followed throughout this work. The
Human Researc h Ethic al Committee of University of B rasilia
Fac ulty of Medic ine approved the experimental protoc ol, and
eac h subjec t examined signed written informed c onsent for partic ipation.
P h a g o c yt i c t e s t . P h a go c yto s i s o f Sa c c h a r o m yc e s c e r e vi s i a e wa s a d a p te d fr o m a te c h n i q u e p r e vi o u s l y
de sc r ib e d3 7. B r ie fly, sample s o f 4 0 ml pe r mar k e d ar e a o f
he par inize d who le pe r iphe r al b lo o d o b taine d b y me ans o f
ve no punc tur e fr o m e ac h sub j e c t we r e plac e d o n c le an glass
slide s c o ntaining 8 mar k e d ar e as o f 7 mm diame te r e ac h, in
duplic ate pr e par atio ns, and inc ub ate d in a we t c hamb e r fo r
4 5 min at 3 7 ° C. The slide s we r e the n r inse d with 0 . 1 5 M
pho sphate -b uffe r e d saline ( PB S) pH 7 .2 at 3 7 ° C to r e mo ve
n o n - a dh e r e n t c e l l s . Afte r wa s h i n g, n e u tr o p h i l s9 2 8 a n d
mo no c yte s3 9 r e maine d adhe r e d o nto the slide appr o ximate ly
in the same pr o po r tio n as the y we r e in the who le b lo o d.
Adhe r e nt c e lls ( 1 2 ,5 3 4 ± 5 ,0 5 0 c e lls/mar k e d ar e a; 5 .6 3 ±
0 . 8 5 % m o n o c yte s a n d 9 3 . 5 ± 1 . 0 8 % n e utr o ph ils ) we r e
inc ubated with a suspension of 2 .5 x1 05 S. c e re visia e in 2 0
µ
lc alf se r um ( FCS) ( Cultilab , Campinas, B r azil) , fo r 3 0 min in
a we t c hamb e r at 3 7 ° C, to allo w phago c yto sis. Slide s we r e
th e n r in s e d with 0 . 1 5 M PB S a t 3 7 ° C to e lim in a te n o n
-phago c yto se d S. c e re vi si a e and the final washing was do ne
with 3 0 % FCS in Ha n k s - tr is . Th e s lide s we r e fixe d with
ab so lute me thano l and staine d with 1 0 % Gie msa so lutio n.
The numb e r o f S. c e re vi si a e phago c yto se d b y 2 0 0 mo no c yte s
o r by 2 0 0 neutr o phils in individual pr epar atio ns was assessed
b y o p ti c a l m i c r o s c o p y. Mi c r o s c o p i c fi e l d s d i s tr i b u te d
th r o u gh o u t th e s l i d e we r e r a n d o m l y s e l e c te d a n d a l l
m o n o c yte s o r n e utr o ph ils in e a c h pa r tic ula r fie ld we r e examined. The phago c ytic index was c alc ulated as the average
numb e r o f phago c yto se d S. c e re vi si a e pe r phago c yto sing
mo no c yte s o r ne utr o phils, multiplie d b y the pe r c e ntage o f
the se c e lls e ngage d in the phago c yto sis3 8.
B aking yeast (Sa c c ha ro m yc e s c e re visia e) was prepared
ac c ording to a tec hnique previously desc ribed3 7. In short, 5 0 g
of fresh live yeast ( Fleisc hmann, Brazil) were resuspended in
2 2 0 ml of PBS, pH 7 .2 , autoc laved at 1 2 0 ° C for 3 0 min, washed
in PBS until obtaining a c lear supernatant and the sediment was resuspended in 2 8 ml of a 0 .1 M 2 -merc aptoethanol solution in
PBS. After 2 hours inc ubation with stirring, yeasts were washed
again and resuspended in 5 5 ml of 0 .0 2 M iodoac etamide in PBS.
Afte r a n e xtr a 2 h o ur s in c ub a tio n with s tir r in g a t r o o m temperature, they were washed 3 times and resuspended in
2 2 0 ml of PBS, pH 7 .2 . Yeasts were again autoc laved, washed
and resuspended in 1 1 0 ml of veronal buffered saline, pH 7 .2 ,
c ontaining sodium azide, and stored at 4 ° C until use. Before
e ac h e xpe r ime nt, de ad S. c e re vi si a e we r e washe d in PB S,
quantified and resuspended in Hanks-tris solution.
Ni t r o b l u e t e t r a z o l i u m s l i d e t e s t . Ni tr o b l u e te tr a zo lium ( NB T) te s t wa s a da pte d fr o m th e te c h n iq ue
de sc r ib e d b y Campb e ll and Do uglas5, aiming to e valuate the
m ic r o b ic idal m e c hanism o f phago c yte s b y the ir ab ility to ge n e r a te to xic o xyge n r a dic a ls c a pa b le o f r e duc in g th e
c o mpo und NB T to an inso lub le fo r m, named fo r mazan, whic h
is ide ntifie d unde r o ptic al mic r o sc o py b y a b lue c o lo r in the c yto plasm o f the c e ll. The amo unt o f NB T r e duc e d is dir e c tly
pr o po r tio nal to the amo unt o f o xyge n r adic als pr o duc e d b y
phago c yte s5, and the se mo le c ule s ar e amo ng the pr inc ipal
m ic r o b ic ida l a ge n ts pr o duc e d b y ph a go c yte s4 9. B r ie fly,
phago c yte s adhe r e d o n slide , as pr e vio usly de sc r ib e d, we r e
inc ub ate d with 0 .0 5 % NB T so lutio n in Hank s-tr is ( Sigma, St
Lo uis, MO, USA) fo r 2 0 min at 3 7 ° C in a humidifie d c hamb e r. The slide we r e the n washe d, fixe d with me thano l and staine d
with a s o lutio n o f 1 . 4 % s a fr a n in a n d 2 8 . 6 % glyc e r o l in distillate wate r. The pe r c e nt phago c yte s with NB T r e duc e d in
the c yto plasm was asse sse d b y o ptic al mic r o sc o py.
Sta tistica l a na lysis. The Kruskal-Wallis non-parametric statistic was used to simultaneously compare the three groups for each variable studied. Multiple comparisons were conducted with
Dunn’s post-hoc test when the Kruskal-Wallis analysis showed an overall signific ant differenc e between the groups. The
Mann-Whitney test was performed to compare two independent groups.
Differences with a two-tailed value of p < 0 .0 5 were considered
statistically significant. The Prism
software package ( GraphPad,USA, 1 9 9 7 ) was utilized for analysis and design of the data.
RESULTS
No statistic ally signific ant differenc e was found between the
median of phagoc ytic index of neutrophils and monoc ytes from
males and females in both c ontrol group and c hagasic individuals
( p > 0 .0 5 ) .
Table 1 shows the c omparison of the different func tional
variables of phagoc ytosis by monoc ytes and neutrophils between the groups of Chagas’ disease patients with and without congestive
heart failure and the healthy c ontrol group.
Alterations in the phagoc ytic func tion of monoc ytes and
neutrophils observed c omparatively between the two groups of patients with Chagas’ heart disease and the c ontrol group are
illustrated in Figure 1 . The group of patients with Chagas’ heart
disease without c ongestive c ardiac failure showed dec reased
monoc yte and neutrophil phagoc ytic func tion as c ompared with
normal subjec ts and c hagasic patients with heart failure.
Ta ble 1- Media n ( extrem e va lues) o f the va ria bles o f in vitro pha go cytic functio n a nd m icro bicida l o xygen ra dica l pro ductio n ca pa city eva lua ted by the nitro blue tetra zo lium reductio n test o f circula ting m o no cytes a nd neutro phils, o bserved fo r co ntro l hea lthy subjects a nd Cha ga s’ hea rt disea se subjects with a nd witho ut co ngestive ca rdia c fa ilure.
Phagocytosis by monocytes Phagocytosis by neutrophils
number of S. cerevisia e % cells engaged phagocytic number of S. cerevisia e % cells engaged phagocytic % NBT phagocytosed in phagocytosis index phagocytosed in phagocytosis index reduction Control subjects 1 .3 2 2 3 .0 2 7 .0 1 .5 8 1 9 .0 2 8 .0 8 6 .5 ( n = 1 1 ) ( 1 .0 0 – 1 .6 5 ) ( 6 .0 – 5 0 .5 ) ( 6 .0 – 8 0 .5 ) ( 1 .1 2 – 2 .2 3 ) ( 3 .0 – 6 8 .5 ) ( 3 .5 – 1 2 5 .0 ) ( 4 5 .5 – 9 8 .5 ) Chagas’ heart disease subjects 1 .1 2 b
4 .0 a,b
4 .0 a,b
1 .2 9 2 .0 a,b
2 .5 a,b
8 1 .5 without cardiac failure ( n = 9 ) ( 1 .0 0 – 1 .4 1 ) ( 1 .5 – 1 6 .5 ) ( 1 .5 – 2 4 .5 ) ( 0 – 1 .9 1 ) ( 0 – 1 8 .5 ) ( 0 – 2 9 .5 ) ( 3 3 .5 – 9 2 .5 ) Chagas’ heart disease subjects 1 .3 3 3 2 .5 4 2 .5 1 .5 6 2 8 .0 4 9 .5 7 4 .7 with cardiac failure ( n = 9 ) ( 1 .0 8 – 1 .7 4 ) ( 2 .0 – 5 4 .0 ) ( 9 .0 – 9 7 .0 ) ( 1 .2 4 – 2 .4 3 ) ( 2 .0 – 4 0 .0 ) ( 3 .0 – 8 8 .5 ) ( 3 4 .5 – 9 4 .0 )
p value * 0 .0 3 0 .0 2 0 .0 0 6 0 .1 7 0 .0 0 3 0 .0 0 3 0 .4 0 * Kruskal-Wallis test. Multiple pair wise comparisons were performed by the Dunn’s post-test when an overall significant difference was verified between the groups.
a significant difference ( p < 0 .0 5 ) between control and Chagas’ heart disease subjects groups by the Dunn’s post-test.
The median of phagocytic index of monocytes of Chagas’ heart
disease patients without heart failure ( 4 ) was several times lower
( 6 .7 and 1 0 .6 , respectively) ( p = 0 .0 0 6 , Kruskal-Wallis test) than
that of control individuals ( 2 7 ) and chagasics with heart failure
( 4 2 .5 ) . No statistically significant difference was observed between
these two latter groups ( p > 0 .0 5 , Dunn’s post-test) . The reduced phagocytic index was due to reduction in the number of phagocytosed
Sa c c ha ro m yc e s c e re visia e and reduc tio n in the number o f monocytes engaged in phagocytosis. Similarly, median of phagocytic
index of neutrophils of Chagas’ heart disease patients without heart
failure was also reduced ( 2 .5 ) ( p = 0 .0 0 3 , Kruskal-Wallis test) in
comparison with normal ( 28) and chagasic with heart failure subjects
( 4 9 .5 ) . This reduc tion was mainly due to lower quantitative
involvement of neutrophils in phagocytosis. Likewise, no statistical
difference was noted between the Chagas’ disease with heart failure
and control groups ( p > 0 .0 5 , Dunn’s post test) .
Patients with Chagas’ heart disease and heart failure showed
no statistically significant differences in the variables of phagocytic
func tion of monoc ytes and neutrophils when c ompared with
c ontrol healthy subjec ts.
The c apac ity to generate toxic oxygen radic als evaluated by
the perc ent reduc tion of NBT was not influenc ed by the presenc e
or absenc e of heart failure in Chagas’ heart disease subjec ts,
and no differenc es were observed between the three groups, as
indic ated in Table 1 and illustrated in Figure 2 .
DISCUSSION
The present study showed that Chagas’ heart disease patients
without c ongestive c ardiac failure presented reduc ed phagoc ytic
c apac ity o f mo no c ytes and neutro phils. On the o ther hand,
patients with heart failure showed phagoc ytic func tion of these
immune c ells similar to healthy subjec ts, suggesting that the
depressed immune func tion of monoc ytes and neutrophils was
reversed in the presenc e of the heart failure syndrome. These Fi gu re 1 - Co m p a ri so n o f m o n o c yte ( le f t) a n d n e u tro p h i l ( ri gh t) p h a go c yti c c a p a c i ty b e twe e n th e gro u p s o f Ch a ga s’ h e a rt d i se a se p a ti e n ts w i th ( n = 9 ) a n d w i th o u t ( n = 9 ) c a rd i a c f a i lu re a n d c o n tro l h e a lth y su b je c ts ( n = 1 1 ) . In d i vi d u a l va lu e s a re sh o wn f o r th e n u m b e r o f Sac c har o myc e s c e r e visiae ( 2 .5 x 1 05 p e r m a rk e d a re a ) p h a go c yto se d b y p h a go c yte ( to p ) , p ro p o rti o n o f p h a go c yte s
e n ga ge d i n p h a go c yto si s ( m i d d le ) , a n d th e re sp e c ti ve p h a go c yti c i n d e x ( b o tto m ) . Th e h o ri zo n ta l li n e s re p re se n t th e m e d i a n va lu e f o r e a c h gro u p . Th e Kru sk a l- Wa lli s a n a lysi s wi th Du n n ’s p o st- h o c te st wa s u se d to c o m p a re th e gro u p s.
N u m b e r o f S . ce re v is ia e in g e s te d b y m o n o c y te s % m o n o c y te s e n g a d e d in p h a g o c y to s is M o n o c y te p h a g o c y ti c i n d e x 3 2 2 1 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0
1 2 5
1 0 0
7 5 5 0 2 5 0 N u m b e r o f S . ce re v is ia e in g e s te d b y n e u tr o p h il s % n e u tr o p h il s e n g a d e d in p h a g o c y to s is N e u tr o p h il P h a g o c y ti c i n d e x 3 2 1 0 7 0 6 0 5 0 4 0 3 0 2 0 1 0 0
1 2 5
1 0 0
7 5
5 0
2 5
0 Contr ol Chagas’ Hear t
Disease
Chagas’ Hear t Disease plus Hear t Failur e
Contr ol Chagas’ Hear t Disease
Chagas’ Hear t Disease plus Hear t Failur e p= 0 .0 3
p= 0 .0 2
p= 0 .0 6
p= 0 .1 7
p= 0 .0 0 3
data suggest that the assoc iation of heart failure syndrome with
c hr o nic Trypa n o so m a c ru zi infec tio n appear s to c ause an
ac tivation of the depressed phagoc ytes in Chagas’ heart disease
patients noted in the absenc e of c ardiac failure.
An enhanc ed ac tivity of mononuc lear phagoc yte c ells has
been demonstrated during ac ute Trypa no so m a cruzi infec tion
in experimental animals6 8 3 1 4 1, whic h was dependent on the
parasite strain ino c ulated in the animals evaluated8. On the
c ontrary, we observed a dec reased phagoc ytic func tion during
chronic cardiac T. cruzi infection without heart failure. Reduction
of chemotaxis of blood monocytes and neutrophils and a tendency
for depressed nitro blue tetrazolium reduc tion by phagoc ytes
has also been observed in patients with c hronic Chagas’ disease
without heart failure1 3 5 0.
The reasons why there were no alterations in phagoc ytic
func tion of monoc ytes and neutrophils in patients with heart
failure assoc iated with c hronic Chagas’ heart disease are not c lear. However, a possible hypothesis to explain our results is
that it was due to an inc reased produc tion of c ytokines c apable of influenc ing phagoc yte func tions when heart failure is present
in patie nts with Chagas’ dise ase . In fac t, alte r e d c yto k ine pro duc tio n has been demo nstrated in patients with Chagas’
disease1 1 and in patients with heart failure syndrome of other
c ause2 3 0 5 3.The c oexistenc e of heart failure and Trypa no so m a
c ru zi infec tion in the patients presently evaluated may have altered the c ytokine sec retion and influenc ed phagoc ytosis. This m ay o c c ur b e c ause the type o f influe nc e o f c yto k ine s o n
phagoc ytosis may depend on c ytokine c onc entration. In fac t, as
we previously observed, TNF-
α
may inc rease or dec rease thep h a go c yti c fu n c ti o n o f m o n o c yte s de p e n di n g o n i ts
c o nc e ntr atio n3 9. In the c o mple x syndr o me o f he ar t failur e
assoc iated with Trypa no so m a cruzi infec tion there are many
fac tors influenc ing the final func tion of eac h c ell. It has been
shown that antigens of Trypa n o so m a c ru zi may induc e the
produc tion of pro-inflammatory c ytokines4, and immune and
inflammatory c ytokines may be inc reased in serum of c hagasic
patients1 1.Plasma c onc entration of inflammatory c ytokines seems
Fi gu re 2 - Co m p a ri so n o f p h a go c yte p ro d u c ti o n o f m i c ro b i c i d a l o x yge n ra d i c a l m o le c u le s a sse sse d a s p e rc e n t re d u c ti o n o f n i tro b lu e te tra z o li u m b e tw e e n th e gro u p s o f Ch a ga s’ h e a rt d i se a se p a ti e n ts w i th ( n = 9 ) a n d w i th o u t ( n = 9 ) c a rd i a c f a i lu re a n d c o n tro l h e a lth y su b je c ts ( n = 1 1 ) . In d i vi d u a l va lu e s a re sh o w n f o r th e p e rc e n t n i tro b lu e te tra zo li u m ( NBT) re d u c ti o n . Th e h o ri zo n ta l li n e s re p re se n t th e m e d i a n va lu e f o r e a c h gro u p . Th e Kru sk a l- Wa lli s a n a lysi s wi th Du n n ’s p o st- h o c te st wa s u se d to c o m p a re th e gro u p s.
to be inc reased in patients with heart failure of any etiology, and
c orrelates with the severity of symptoms1 7 2 9. On the other hand,
heart failure is assoc iated with hemodynamic , hydro-elec trolytic ,
hormonal and autonomic neural disturbanc es and may also
modify the c ytokine produc tion. Thus, it c an be c onjec tured that
the interplay between these func tional alterations resulting in c ounterbalanc ed opposite immune effec ts is the probable c ause
fo r th e pa r a do xic a lly n o r m a lize d ph a go c ytic c a pa c ity o f
phago c ytes o bser ved in Chagas’ hear t disease patients with
c ongestive heart failure, although the phagoc ytic func tion of these
immune c ells is ac tually defic ient pe r se .
An alternative hypothesis to explain the differing phagoc ytic
func tion observed would be a different deregulation of T c ell
regulatory sec reting IL-1 0 or TGF-
β
in patients with Chagas’disease in presenc e or absenc e of heart failure3 5.
It should be also c onsidered the possibility of the influenc e on phagoc ytosis by neutrophils and monoc ytes of some drugs in
use by the patients with heart failure. However, it seems that this
was not the case in the present study, since it has been shown that
furosemide and spironolactone does not alter phagocytosis2 3 3 6,
and c apto pr il, e nalapr il, am io dar o ne o r hydr alazine m ay
dec rease phagoc yte func tions1 4 1 5 4 0 4 5 c ausing an opposite effec t
of that observed in the patients with heart failure presently studied.
The capacity to reduce NBT to formazan indicates that the
phagocytes are producing oxygen radicals, which are important
microbicidal mechanism of these cells causing pathogen death5 4 9.
Unlike the o bser vatio n o f dec r eased phago c ytic c apac ity o f
monoc ytes and neutrophils in Chagas’ heart disease patients
without heart failure, we were unable to detec t any alteration in
perc ent reduc tion of NBT. This fac t suggests that the destruc tion
of pathogens after ingestion by phagoc ytes was not c ompromised
in the patients examined with or without heart failure. On the
c ontrary to the present findings, Voltarelli e t a l5 0 observed a
tendenc y for diminished perc ent of NBT reduc tion in c hagasic
patients with cardiac disease without heart failure or with digestive
form of the disease. These results apparently c onflic t with ours,
and may be related to different c linic al aspec ts or stages of
evolution of Chagas’ c ardiopathy in the subjec ts in eac h study.
Many unfavorable c onsequenc es, suc h as predisposition
to infec tions or direc t mec hanic al heart dysfunc tion, c an result
from partially inadequate func tion of phagoc ytes in c hagasic
patients, c onsidering that these immune c ells play multiple roles
i n r e s i s ta n c e m e di a te d b y th e i m m u n e s ys te m a ga i n s t Tr yp a n o s o m a c r u z i2 5 3 1. I t s h o uld b e s tr e s s e d th a t th e
normalized phagoc ytic func tion noted in Chagas’ heart disease
a sso c ia te d with c a r dia c fa ilur e se e m s to b e a se c o nda r y
ph e n o m e n o n o c c ur r in g po s s ib ly a s a b a la n c e d e ffe c t in
c onsequenc e of inc reased levels of c ytokines enc ountered in
non-spec ific heart failure2 3 0, whic h stimulate the phagoc ytes.
Neutrophils and monoc ytes are also involved in first line defense
against infec tions by extra-c ellular bac teria1 8, and patients with
heart failure may present different infec tions that threaten their
lives, as a c o mplic atio n c aused by bac teria that depend o n
normal phagoc ytes func tion for their elimination3, suggesting
dysfunc tion of these c ells. Contr ol Chagas’ Hear t
Disease
Chagas’ Hear t Disease plus Hear t Failur e
%
N
it
r
o
b
lu
e
t
e
tr
a
z
o
li
u
m
r
e
d
u
c
ti
o
n
b
y
p
h
a
g
o
c
y
te
s
1 2 5
1 0 0
7 5
5 0
2 5
0
One possible limitation of the present study c onc erns the
extrapolation of an in vitro laboratory finding for a clinical condition
that involves a multiplicity of interplaying functional factors. In this
respect, it should be pointed out that there is no alternative means to
that utilized here to demonstrate the effects of different factors acting
directly on phagocytic cells. It is obvious that the definitive proof of the in vivo relationship between the phagocytic function of monocytes
and neutrophils and the occurrence or predisposition or not to
infec tions in patients with or without heart failure requires a
prospectively designed longitudinal clinical study.
We b e lie ve th a t o ur s tudy un de r s c o r e s th e c o m ple x pathophysiologic al interplay between Chagas’ heart disease, the
c onsequent heart failure syndrome and phagoc yte func tions,
showing that the immune system may be differently c ompromised
in both c linic al c onditions ac c ording to the heart func tional status, whic h suggests that the disturbanc es pro per to eac h
c ondition pe r se appear to affec t one another. In the c linic al
setting, the resulting immune alterations of these interplaying
patho physio lo gic al me c hanisms may influe nc e the c linic al evolution and the outc ome of the affec ted patients.
In c onc lusion, our data showed that phagoc ytic func tion of
mo no c yte s and ne utr o phils was c o nside r ab ly de c r e ase d in
c hronic Chagas’ c ardiopathy in the absenc e of heart failure, while
this func tion was observed to be normal in the presenc e of the c ongestive syndrome. This fac t suggests that balanc ed opposite
effec ts on the phagoc ytosis, c onsequent to c ardiovasc ular and
immune disturbanc es, may be ac ting in Chagas’ c ongestive heart
failure and paradoxic ally normalizing the phagoc ytic func tion. The altered phagocytic function of Chagas’ disease patients should
be taken into c onsideration when it is nec essary to presc ribe
drug therapy, suc h as for example antibiotic s, that may also
influenc e phagoc yte func tions, in order to avoid aggravation of these defic ienc ies.
ACKNOWLEDGEMENTS
The authors gratefully thank Mrs. Renê Oliveira Pires and Mr. José Siqueira for technical assistance. Dr. Lícia Maria Mota and Dr. Rodrigo Barbosa Aires were fellowship recipients supported by PIBIC-UnB/CNPq at the Laboratory of Cellular Immunology.
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I n c r e a s e d l e ve l s o f s e r u m n e o p te r i n a n d de c r e a s e d p r o du c ti o n o f ne utr o phil supe r o xide anio ns in c hr o nic he ar t failur e with e le vate d le ve ls o f tumo r ne c r o sis fac to r-α. J o ur nal o f the Ame r ic an Co lle ge o f Car dio lo gy 2 2 : 1 8 9 7 -1 9 0 1 , 1 9 9 3 .
