Luciana M Silva, André LM Fernandes, Aryon Barbosa Jr, Irismar R Oliveira, Zilton A Andrade"*"
Centro de Pesquisas Gonçalo Moniz-Fiocruz, Rua Valdemar Falcão 121, 40295-001 Salvador, BA, Brasil H e p a tic S c h isto so m a m anson i p e r io v u la r g ra n u lo m a s u n dergo ch a n g e s in size, c e llu la r c o m p o si
tion a n d a p p e a r a n c e w ith tim e. This p h en o m en o m , know n as “im m u n o lo g ic a l m odu lation ”, h as been th ough t to re fle c t h o st im m u n o lo g ic a l sta tu s. H ow ever, a s m o d u la tio n h as n o t been o b s e r v e d o u tsid e the liver, p a r tic ip a tio n o f lo c a l f a c to r s , h ith erto little con sid ered , se e m s cru cial. C o m p o n en ts o f the ex tra c e llu la r m a trix o f p e r io v u la r g ra n u lo m a s o f the m ou se w e re p a r tic u la r ly stu d ie d in three different o r g a n s (liver, lu n g a n d in testin e) a n d d u rin g th ree p e r io d s o f infection tim e (acute, in term ed ia te an d chronic) b y m e a n s o f h isto lo g ic a l, b io c h e m ic a l a n d im u n oflu orescen ce tech n iqu es, w h ile q u a n tita tiv e d a ta w ere e v a lu a te d b y c o m p u te r iz e d m o rp h o m etry, in o r d e r to in v e stig a te p a r tic ip a tio n o f lo c a l fa c to r s in g ra n u lo m a m od u la tio n . R e su lts c o n firm e d m o d u la tio n a s a e x c lu siv e ly h e p a tic p h en om en om , sin ce p u lm o n a ry a n d in te s tin a l g ra n u lo m a s, f o r m e d a ro u n d m atu re eggs, d id n o t ch a n g e s iz e a n d a p p e a ra n c e with tim e. The m a tr ic ia l c o m p o n e n ts w h ich w e re in ve stig a te d (Type I, III a n d I V colla g en s,fib ro n ectin , laminin, p r o te o g ly c a n s a n d e la stin ) w e re f o u n d in a ll g ra n u lo m a s a n d in a ll o rg a n s exam ined. H ow ever, th eir p r e s e n c e w a s m uch m o re p r o m in e n t in th e liv e r E lastin w a s o n ly f o u n d in h e p a tic g ra n u lo m a s o f ch ron ic in fection. The la rg e a m o u n t o f e x tr a c e llu la r m a trix c o m p o n e n ts f o u n d in h ep a tic g ra n u lo m a s w a s the m ain c h a n g e r e sp o n sib le f o r the m o r p h o lo g ic a l a sp e c ts o f m o d id a tio n . Therefore, the p e c u lia r en viron m en t o f th e liv e r u ltim a te ly d e te rm in e s th e ch a n g es id e n tifie d in sc h is to so m a l g ra n u lo m a as “m odu la
tion ”,
Key words: modulation - granuloma - shistosomiasis - extracellular matrix
M e m In s t O s w a ld o C ruz, Rio d e Ja n e iro , Vol. 9 5 (3 ): 3 5 3 -3 6 1 , M ay/Jun. 2 0 0 0 353
H ep a tic g ra n u lo m a s form ed around m ature e g g s o f S c h isto so m a m a n so n i undergo ch a n g es in size, appearance and cellular constitution w ith time.
T h is has b een interpreted as a m ech an ism o f host p rotection, w ith the h ost reactions b eco m in g m ore e c o n o m ic a l and e ffic ie n t during chronic in fection (A ndrade & Warren 1964). T h e im m u n o lo g ica l counterpart has b een thorough ly in vestigated , both in v iv o (C o lle y 1981, C hensu e et al. 1993, B o g e n et al. 1995) and in vitro (D o u g h ty & P h illip s 1982, Parra et al. 1 991), in clu d in g its cy to k in e patterns (W ynn & C h eev er 1995, M cK errow 1997). H o w ever, there are data in d icatin g that gran u lom as ou tside the liver m ay b eh ave differently. G ranulo
m as in the in testine w ere ob served to be already m odulated in the ileu m from the very b egin n in g, although m od u lation w a s said to occur in the c o lon and ileal P e y e r ’s patch (W ein stock & B oros 1981, 1983). In the lu n gs granulom as p resented
Supported by Papes 2 (Fiocruz) and Pronex.
^Corresponding author. Fax; *55.71.356.4292. E-mail:
[email protected] Received 21 May 1999 Accepted 13 January 2000
the sam e m orp h ology regardless the tim e o f in fe c
tion (Vidal et al. 1992, E ltou m et al. 1995). Such find in gs su g g est that the so -ca lled “im m u n ologi
cal m odulation” o f periovular granulom as in sc h is
to so m ia sis is m ainly in flu en ced by local factors.
I f this is so , the current interpretation o f the im m u n ological m odu lation con cept needs to be re
vised.
To further in vestigate this p ossib ility, a co m parative study o f siz e, appearance and matrix com p osition o f granulom as form ed in three different organs o f m ice (liver, intestine and lung), during three different p eriods o f in fection (early, interm e
diate and late), w a s undertaken. M ethods o f histo- pathology, im m u n oflu orescence, biochem istry and m orphom etry w ere applied in an attempt to under
stand the real sig n ifica n ce o f the so -ca lled “im m u n ological m odulation” o f schistosom al granuloma.
MATERIALS ANT) METHODS
T hrity-one albino S w iss m ice o f both sex es, w eig h in g 18-23 g, m aintained w ith free access to a com m ercial balanced diet and water, w ere used.
A n im als w ere subm itted to transcutaneous in fec
tion w ith 5 0 recently elim inated S. m a n so n i cer- cariae each. Later, anim als w ere random ly divided into three groups, accord in g to tim e o f infection;
first group (acute p h ase), anim als sacrificed eight
3 5 4 G r a n u lo m a M o d u la tio n • L uciana M Silva e t al.
w ee k s fo llo w in g cercarial exposure; se co n d group (interm ediate phase), anim als sacrificed at the 16th w ee k after ex p o sitio n ; and third group (ch ron ic phase), anim als sacrificed at the 22n d w e e k s o f infection.
To augm ent the ch a n ces o f fin d in g pulm onary granulom as, an extra group o f m ice w a s in fected with 100 cercariae. F iv e anim als w ere in clu d ed in the acute phase group, and another fiv e in th e in
term ediate group. N o an im als in fected w ith 100 cercariae su rvived to b e in clu d ed in the ch ron ic group.
/’ A t the tim e o f sa crifice the an im als w ere an
es-! thetized w ith ether, the ab d om en w a s op en ed and V the aorta se v er ed . T h e liv e r w a s r e m o v e d and
w eigh ed . T h e cou n tin g o f w orm s w a s perform ed by exam in in g the m esen teric v ein s and b y sm a sh ing the liver b etw een tw o slid e s. In this last ca se, several fragm ents to be subm itted to other tec h niques w ere p rev io u sly saved . A lo b e o f the lu n g w as fixed by intra-tracheal in jection o f 10% for
m alin. Fragm ents o f the liver, sm all in testin e and lung w ere u sed for the fo llo w in g purposes:
H is to lo g y - T issu e s w ere fix ed in either pH 7 .2 phosphate b u ffered 10% form alin in P B S or in B o u in ’s fluid. T h e 5(x-thick paraffin se c tio n s o b tained w ere stain ed w ith the fo llo w in g m ethods;
h em a to x y lin and e o s in , siriu s-red for c o lla g e n , G om ori’ s silv er im pregnation for reticu lu m and A lcian -b lu e stain ing under tw o differen t p H s (2 and 7).
C o u n tin g o f e g g s - Fragm ents o f the liv e r and intestines w ere w eig h te d and d igested overn igh t in 4% p otassiu m h yd roxid e and the e g g s cou n ted accord in g to C h e e v e r ’s m eth od (1 9 7 0 ). R esu lts w ere exp ressed as con centration o f e g g s per gram o f tissu e.
Im m u n o flu o rescen ce - Fragm ents o f liver, in
testines and lu ng w ere p laced in sm all carton b o x es filled w ith T issu e-T ek (M ile s, U S A ) and im m e d i
ately frozen in liquid n itrogen for 5 m in. B lo c k s w ere then k ept in air-thight p lastic b o ttles at -8 0 °C until sectio n ed in a criostat at -2 0 °C . T he se ctio n s w ere co llec te d on clean g la ss slid e s and im m e d i
ately w ash ed in iced salin e and treated w ith either one o f the fo llo w in g anti-sera; a n ti-collagen I, anti
collagen III, an ti-collagen IV, anti-fibronectin, anti- lam inin, and anti-desm in. A ll anti-sera w ere k in d ly supplied by Dr Jea n -A lex is Grim aud. T he anti-sera w ere derived from hum an m aterial and w ere pre
pared in rabbits. T h eir d ilu tion at the m om en t o f use varied from 1:50 and 1; 100. D eta ils about their preparation and sp ec ificity can b e found elsew h ere (Andrade & G rim aud 1986). T h e secon d ary anti
b o d y w a s a f l u o r e s c e i n a t e d a n ti- r a b b it Ig G (Sigm a), diluted at 1 ;40 in a w eak solu tion o f E vans bjue., j^To/itrols co n siste d o f se ctio n s treated w ith
saline o n ly and se c tio n s treated w ith an irrelevant serum (norm al rat serum ).
H y d ro x y p ro lin e m e a su rem e n ts - Fragm ents o f the liver w ere w e ig h e d and then subm itted to b io ch em ical d eterm in ation o f hydroxyprolin e content b y th e c o lo r im e tr ic m eth o d B o f B ergm an and L o x le y (1 9 6 3 ). S a m p les w e ig h in g from 100 to 25 0 m g w ere used . H yd roxyp rolin e le v e ls w ere cor
rected for in ten sity o f in fectio n b y d iv id in g total h ep a tic h y d r o x y p r o lin e (w ith o u t co rrectio n for h yd roxyp rolin e le v e ls in u n in fected m ic e ) b y the num ber o f S. m a n so n i e g g s in the liver.
M o r p h o m e tr y - H isto lo g ic a l se c tio n s either stain ed w ith h em a to x y lin and eo sin or sirius-red w ere an alyzed b y autom atic m orphom etry u sin g a L e ic a Q 5 0 0 M C Im age P ro c essin g and A n a ly sis S ystem (L eica C am bridge, C am bridge, England).
For m orp hom etric m easu rem ents a total sectional area o f 17.01 xlO ^ )im^ per case w a s evaluated (C ou tin h o et al. 1997).
S ta tistic a l a n a ly sis - N um erical v alu es obtained from e g g co u n tin g , h yd roxyp rolin e m easurem ent and m orop hom etric data w ere analyzed u sin g para
m etric tests. A n a ly sis o f variance w ith on e or tw o factors (A n o v a ) w a s u sed for iden tification o f s ig nifican ce. S h id en t-N ew m an -K eu ls and the Student- t tests w er e ap p lied for group d ifferen ces. R esu lts revealin g p < 0 .0 5 w ere con sid ered sign ifican t.
RESULTS
A n average o f fiv e w orm pairs per m ou se w as recovered , and that decreased to three during the interm ediate and chronic phases o f infection. W hen 100 cercariae w er e u sed , tw ic e as m any w orm s w ere cou n ted. T h e num ber o f e g g s p er gram o f tissu e w a s greater in the liver ( 1 4 ,7 1 3 ) than in the intestines (8 ,4 5 1 ), this d ifference b ein g statistically sig n ifica n t (p < 0 ,0 5 ). H ow ever, w h en m orp hom et
ric m easu rem en ts for num erical d en sity w ere c o n sidered, the v a lu e s for the intestine w ere greater than th o se for the liver (Table I). M orphom etry also sh o w e d that th e v o lu m e o f the granulom as, c o n sid ered as b e in g sp herical, d ecreased sig n ifica n tly in the liver, e s p e c ia lly w hen th ose from the acute phase w ere com p ared w ith the chronic p hase. N o sig n ifica n t d iffer en ce s in granulom a v o lu m e w ere n oted for th o se form ed in the in testin e or lu n g (Table II). R egardin g v o lu m e density, w h ich m eans the total v o lu m e occu p ied by the granulom as w ithin a g iv e n unit o f v o lu m e, the v a lu es for the liver w ere greater during the acute p h ase, d ecreasin g th ereaf
ter. S u ch d ecrease did not occur for the in testine or lung. D urin g th e acute p hase, granulom a v o lu m e d en sity for the liv e r w a s greater than for the in tes
tin e an d lu n g , but during the in term ed iate and chronic p h a se s such d ifferen ce lack ed statistical sig n ifica n ce (T able III).
Values for numerical density (Nn) o f granulomas in different organs o f m ice infected with 50 Schistosoma mansoni cercariae during three periods o f time, seven animals in each group
M e m ¡n s t O s w a ld o C ru z, Rio d e J a n e iro , Vol. 9 5 (3 ), M ay/Jun. 2 0 0 0 3 5 5
T A B L E I
Organs Time o f infection (Nn x nm^)
8 weeks 16 weeks 22 weeks
Lungs 1 ,3 6 = 1,78 3,29 = 4,35 5,38 ± 5 ,7 7
Liver 12,24 = 2,09 14,89 ± 2,89 18,53 ±4,29^
Intestines 15,59 ± 2,96 16,48 ± 2 ,4 2 21,93 ± 7,2 1 '’
a\ P< 0,05 (Student-Newman Keuis) as compared to 8 weeks; b: p<G,05(Student-Newman Keuls) as compared to 8 and 16 weeks.
TABLE II
A\ erage values for granuloma volume in different organs and at different periods o f time in m ice infected with 50 Schistosoma mansoni cercariae, seven animals in e ach group________________________
Organs Time o f infection (x 10"* |im^)
8 weeks 16 weeks 22 weeks
Lungs Liver Intestines
17,62 = 27,62 481,53 =161,51 22,96 = 15,13
44,28 ± 43,23 252,98 ± 227,66
46,71 = 17,13
32,56 ± 3 9 ,1 9 84,32 ± 26,22“^
16,96 ± 4,49 a: p<0,05(Student-Newman Keuls) as compared to 8 and 16 weeks.
TABLE ni
\a lu es for volume density (Vv) o f periovular granulomas in different organs and at different periods o f time in mice infected with 50 Schistosoma mansoni cercariae, seven animals in each group
Organs Time o f infection
8 weeks 16 weeks 22 weeks
Lungs 0,00 ± 0,00 0,01 ± 0 ,0 1 0,01 ± 0 ,0 2
Liver 0,18 ± 0 ,0 5 0,012 ± 0 ,0 5 0,07 ± 0,02“^
Intestines 0,04 ± 0,02 0,09 ± 0,03a 0,04 ± 0,03
a: p<0.05(Student-Newman Keuls).
Histological evaluation was concerned only with periovular granulomas having a well pre- ser\ ed central miracidium. Old, involuting granu
lomas were not considered. Granulomas were abundant in sections from the liver. During the acute stage they appeared as predominantely exu
dative, frequently exhibiting a central halo of ne
crosis and a sprinkling of polymorphonuclear eosi
nophils (Fig. 1). Collagen fibers were thin and loosely arranged in a concentric disposition. Un
der immunofluorescence microscopy, Type III col
lagen appeared more abundant than type I, this latter being represented by fibers that seemed thicker and straight, rather than wavy, when com
pared with Type III collagen (Fig. 7A, B).
Fibronectin formed a prominent network of fine fibrils all over the granuloma area (Fig. 1C). Type FV' collagen and laminin were absent from perio- Milar granulomas, revealing only basement mem
branes in small blood vessels situated at the granu
loma periphery (Fig. 7D). A pale, diffuse Alcian blue staining marked all granulomas. This stain
ing was similarly observed with either acidic and neutral pH. Within the structoires of the miracidia, the Alcian blue staining became more accentuated, especially when a neutral pH was employed.
During the intermediate phase hepatic granu
lomas differed from those of the acute phase in showing a tendency to confluence and in being smaller and with the cells and collagen fibers be
coming more packed. Eosinophils were less nu
merous while macrophages and fibroblasts became more prominent.
At the chronic stage periovular granulomas were especially characterized by being smaller, well delimited and more fibrotic than before (Fig.
2). Collagen fibers were thicker, concentrically distributed, and strongly fluorescent when tested
3 5 6 G ra n u lo m a M o d u la tio n • Luciana M Silva et al.
M e m In s t O s w a ld o C ru z, Rio d e J a n e iro , Vol. 9 5 (3 ), M ay /Ju n . 2 0 0 0 3 5 7
for the presence of type I and type III collagens.
The staining for fibronectin was less intense than that obtained for the acute phase. Orcein-positive elastic fibers, conspicously absent in acute and in
termediate phase granulomas, appeared in a few granulomas of the chronic phase (Fig. 3). Desmin- positive cells increased their numbers proportion
ally from acute to chronic phase.
Granulomas formed around mature eggs in the intestine and lung showed a mixture of proliferative and exudative features, but they tended to maintain the same morphologic aspect when examined at dif
ferent times of infection (Figs 4,5). All components of extracellular matrix revealed by immunofluores
cence microscopy in the liver were also present in granulomas of intestine and lung, although always in smaller amount (Fig. 7E, F).
Hydroxyproline content was evaluated only for the liver. Data expressed as jimol of hydroxypro
line per gram of tissue versus number of eggs per gram of liver are depicted in Fig. 6A, B, C. Posi
tive correlation occurred at the acute phase only.
However, the concentration of hydroxyproline in the liver increased progressively with time of in
fection. The average numbers were: normal liver 3.42 jimol; acute phase 6.90 jimol, intermediate phase 7.84 )imol and chronic phase 8.77 |imol.
DISCUSSION
Present findings confirm and extend previous data which are suggestive that the so-called “immu
nological modulation” of schistosomal periovular granuloma is a morphological phenomenom pecu
liar to the liver (Grimaud et al. 1987, Vidal et al.
1992). Granulomas formed around eggs injected directly into the lungs (Lichtenberg 1962) or liver (Edungbola & Schiller 1979) differ accordingly with the previous immunological state of the host, that is, whether the recipient animal is already S. mansoni infected or not. However, injections of eggs into normal animals or even into those already infected (Domingo & Warren 1968), create an artificial and complex situation, with some peculiar features that differ from those of a cercarial infection, as has been discussed by Cheever et al. (1998).
Weinstock and Boros (1981, 1983) observed differences in size and cellular composition of granulomas formed at different intestinal segments.
Probably these differences were small. Present data refer to the intestines as a whole, and no evidences of modulation were noted in that organ during dif
ferent periods of infection.
Quantitative data about granuloma size and volume were subjected to morphometric
evalúa-Fig. 3; a well delim ited inflam m atory reaction around a schistosom e egg form ed during the chronic phase o f infection, exhibits fme, concentric, black, orcein-positive elastic fibers. O rcein m ethod for elastic fibers, 200X.
-w.. '
3 5 8 G r a n u lo m a M o d u la tio n • L u cian a M Silva e t al.
Fig 4: a mixture o f exudative and proliferative features characterizes periovular granulom as form ed w ithin the pulm onary alveolar tissue. Intermediate phase o f infection. H & E, 200X.
Fig 5: a small granulom a form ed around a m ature Schistosom a m ansoni egg in the intestinal subm ucosa, during the acute period o f infection. H & E, 200X.
■ ijsr-. »•
M e m In s t O s w a ld o C ruz, Rio d e J a n e iro , Vol. 9 5 (3 ), M ay/Jun. 2 0 0 0 3 5 9
Fig 6: correlation between the num ber o f eggs and hydroxypro- line content in the liver o f mice infected with 50 Schistosoma mansoni cercariae at different periods o f infection. A; acute phase (8 weeks); B: intermediate phase (16 weeks); C: chronic phase (22 weeks),
•A,...'
tion, and the nature o f granulom atous extra cellu lar m atrix w a s an a ly zed b y im m u n o flu o rescen t m icroscopy. R esu lts indicated that ch an ges, or lack o f it, regarding siz e , appearance and cellu lar co m p o sition o f granulom as located at different organs and at different tim e s w o u ld m o stly d ep en d on lo cal factors. T h e in flu en ce o f general factors, such as im m une c e lls , cytok in e patterns and so on, is ce rta in ly im portan t. H o w e v e r , m ore atten tion sh ou ld b e paid to the p ecu liar cellular p opu lation o f the liver and the extracellular m atrix it can pro
duce and degrade. T he m atricial com p onents w hich w ere in vestigated (T ype I, III and IV co lla g en s, fibronectin, lam in in and p ro teo g ly ca n s) w ere also found in all gran u lom as and in all organs exam ined. H o w ev e r, their p resen ce w a s m u ch m ore p rom inent in the liver. A s a m atter o f fact, elastic fibers (elastin ) w ere found on ly in h ep atic granu
lom as, and e x c lu siv e ly during the chronic stage o f in fection , but that fin d in g w a s the on ly on e w h ich w a s q u alitatively d ifferen t from the granulom as form ed elsew h er e. A lth o u g h ela stic fib ers w ere abundant in portal sp ace fibrosis due to sc h is to so m iasis in m an, p eriovular granulom as w ere said to be d evoid o f su ch fibers (Junqueira et al. 1986, A ndrade & Freitas 1991). A n yw ay, quantitatively, the large am ount o f extracellular m atrix found in hepatic granulom as w a s the m ain ch an ge resp on sib le for the m o rp h ologic asp ects o f m odulation.
Probably, the p resen ce o f sp ecial cellular elem en ts, such as, h ep atocytes, Ito c e lls and K u p ffer cells, w hich are k n ow n to coordinately p lay an im por
tant role in form ation and degradation o f extracel
lular m atrix in the liver (B lo m h o ff & W ake 1991, Casu et al. 1994, G ressner & B ach em 1994, Enzan et al. 1995) accou n ts for the p ecu liar w a y p erio v u lar granulom as are m odulated in that organ.
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