R e v is ta d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 7 ( 4 ) : 2 2 1 - 2 2 6 , o u t- d e z , 1 9 94.
ASPECTS OF THE GRANULOMATOUS REACTION IN THE
LIVER OF MICE INFECTED AND REINFECTED WITH TWO
DIFFERENT GEOGRAPHICAL STRAINS OF
S C H IS T O S O M A M A N S O N 1P ed ro R aso, P aulo M arcos Z . Coelho, N ivaldo H . T o p p a an d R ôm ulo T . Mello
In th is s tu d y , w h ic h w a s u n d e r ta k en in re la tio n to th e h is to p a th o lo g ic b e h a v io r o f tw o d iffe r e n t s tr a in s ( L E - B e lo H o riz o n te , M G a n d S J - S ã o J o s é d o s C a m p o s , S P ) in in fe c tio n s a n d r e in fe c tio n s (h om olo g ou s o r h e te r o lo g o u s ) w ith Schistosoma mansoni, th e a u th o r s c o n fir m e d a m o r e a c c e n tu a te d p a th o g e n ic ity o f th e S J s tra in . A l l th e r e in fe c tio n s s h o w e d th e p r e s e n c e o f ty p ic a l g r a n u lo m a s o fth e a c u te p h a s e , w h en p e r fo r m e d eith e r w ith th e s a m e s tr a in (h o m o lo go us ) o r w ith a d iffe r e n t s tr a in (h eter olo g ou s) o f the p a r a s ite o f th e p r im o in fection . The p o s s ib le m e c h a n is m s r e s p o n s ib le f o r r e a ctiv a tio n o f th e im m u n o p a th o lo g ic r e s p o n s e in r e in fe c tio n s a r e d is c u s s e d .
K e y -w o r d s : G ra n u lo m a . G ra n u lo m a to u s re a ctio n. Schistosoma mansoni. G e o g r a p h ic a l s tr a in s.
Though recent investigations have elucidated some aspects o f the interdependence o f local reactions against evolutive forms o f S c h is to s o m a m a n s o n i r e la te d to th e c lin ic a l fo rm o f schistosomiasis, further work is needed to clarify the interrelations that command and regulate the appearance o f the various hepato-splenic forms o f the disease m ore comprehensively. Aiming at explaining the hepato-splenic forms o f the disease, it is im portant to consider some factors related to the host such as immunological response, age, organ prom inently affected, etc., several intim ately connected w ith the parasite must be taken into account, as the p arasite’s strain, intensity o f infection, and num ber o f reinfections with the same or different strains. W hile our former work showed that the SJ strain o f S . m a n s o n i caused severer granulom atous lesions around eggs than the ones elicited by the LE strain5, the present study aims to
U n iv e rsid a d e F e d e ra l de O u ro P re to , O u ro P re to , M G e D e p a rta m e n to s d e P a ra s ito lo g ia d o In stitu to d e C iê n c ia s B io ló g ic a s, d e A n a to m ia P ato ló g ica da F a c u ld a d e de M e d icin a e d e A nálises C lín icas e T o x ic o ló g ica s d a F aculdade de Farm ácia, U n iv e rsid a d e F e d e ra l d e M in a s G e ra is, M G , B rasil. F in a n c ia l su p p o rt fro m C N P q , F A P E M IG , F IN E P , P R P q , B razil a n d W H O , S w itz e rla n d .
A d d r e s s to '. P ro f. P a u lo M a rc o s Z . C o e lh o . G ID E /IC B /U F M G , C a ix a P o sta l 4 8 6 , 3 0 1 6 1 -9 7 0 B elo H o riz o n te , M G , B rasil. R e c e b id o p a ra p u b lic a ç ã o e m 1 0 /0 3 /9 4 .
determine whether reinfection performed in mice with both strains would result in new acute phases o f the disease.
M ATERIALS AND M ETHODS
S c h is to s o m a m a n s o n i (L E a n d S J strain s)
LE strain. This strain was isolated from feces o f a patient living in the region o f Belo H orizonte, and kept in B io m p h a la r ia g la b r a t a for more than 30 years at the laboratories o f the Schistosomiasis R esearchUnit, Federal University o f M inas Gerais, Brazil.
SJ strain. It was isolated from field B io m p h a la r ia te n a g o p h ila snails coming from the region o f São José dos Campos, Paraíba Valley, State o f São Paulo, Brazil, and maintained by laboratory passage in B . g la b r a ta andB . t e n a g o p h ila , alternatively, at the Schistosomiasis Research U nit, for m ore than
15 years.
R a s o P , C o e lh o P M Z , T o p p a N H , M e l lo R T . A s p e c t s o f th e g r a n u l o m a t o u s r e a c t io n in th e l i v e r o f m i c e in f e c te d
a n d r e i n f e c t e d w ith t w o d if f e r e n t g e o g r a p h i c a l s t r a in s o /S c h is t o s o m a m a n so n i. R e v i s t a d a S o c i e d a d e B r a s i l e i r a d e M e d i c in a T r o p i c a l 2 7 : 2 2 1 - 2 2 6 , o u t- d e z , 1 9 9 4 .
A fterw ards, the groups w ere divided as follows:
group I - M ice infected w ith the LE strain,
group II - M ice infected w ith the SJ strain, group III - M ice infected w ith the LE strain and
reinfected w ith the same strain, group IV - M ice infected w ith the SJ strain and
reinfected w ith the same strain, group V - M ice infected w ith the LE strain and
reinfected w ith the SJ strain,
group VI - M ice infected w ith the SJ strain and reinfected w ith the LE strain, group V II - Control mice (non-infected).
T he animals w ere sacrificed at 150 days after prim o infection, that is, 60 days after the last infection, and their livers w ere removed. From each liver, a 3.0m m thickness slices were formalin- fixed and paraffin-em bedded. F o r histological exam ination tissue sections w ere stained w ith hem atoxylin and eosin (HGE) and Sirius Supra-Red F3 BA9, and examined using a Zeiss Microscope w ith polarized light.
RESULTS
G roup I - As regards the livers pertaining to this group, w hich received a single infection w ith cercariae (LE strain), and whose animals were sacrificed 150 days later, the m icroscopic features w ere id en tical. T he fundam ental lesion was represented by granulom atous reaction around eggs scattered in the portal tracts and parenchyma. The granulom as w ere found to be coetaneous, thus indicating the same origin related to ju s t one egg- laying o r various ones very close to one another. All o f them evidenced (through hem atoxylin and eosin staining) the characteristic aspect o f the 3rd and 4* evolutive phases, that is, productive phase o r in process o f cure by fibrosis, both o f them denoting the chronicity o f infection. The granulomas were sm all-sized, w ith little exudation surrounding eggs, and presented irregular fibrosis clearly.
There was no significant cuffing in the periportal connective tissue, w hich contained m ild infiltrate o f eosinophils, lymphocytes and plasma cells.
The hepatocytes showed m ild degenerative
phenom ena (loss o f basophilia, hydropic o r
vacuolar degeneration) and severe polyploidy. The sinusoids revealed a moderate congestion and dilation. The Kupffer cells w ere hypertrophic and h y p e rp la s ic an d c o n ta in e d s o m e tim e s schistosome pigment.
Under polarized light the collagen fibrillae appeared in most granulomas in the form o f irregular fibrillae, w ith variable thickness, generally short sized, intercrossed and oriented in various directions, so as to confer them a “b ird ’s nest” aspect (Figure 1). Unusually, the collagen fibrillae tended to form arched sets o f parallel fibers, superposed like a onion skin appearance.
F igu re 1 - A sm a llg r a n u lo m a fr o m g r o u p III (sta in e d by
S iriu s S u p ra R e d ) sh o w in g an a s p e c t s im ila r to a " bird's n est" d u e to p a r a lle lfx b e r s .
R a s o P, C o e lh o P M Z , T o p p a N H , M e llo R T . A s p e c ts o f th e g r a n u lo m a to u s r e a c tio n in th e liv e r o f m ic e in fe c te d a n d r e in fe c te d w ith tw o d if fe r e n t g e o g r a p h ic a l s tr a in s o/gchistosom a mansoni. R e v is ta d a S o c ie d a d e B r a s il e ir a d e M e d ic in a T r o p ic a l 2 7 :2 2 1 -2 2 6 , o u t-d e z , 1 9 94.
d) the degenerative phenomena of the hepatocytes and polyploidy w ere more severe; e) the dilated and congested sinusoids showed hypertrophic and hyperplasic Kupffer cells, with a large amount o f schistosome pigment.
Observation o f the granulomas (under polarized light) revealed the same aspect above mentioned. As occurred on the animals from group I, no worms or lesions caused by them could be seen in this group.
Based on this comparative result, it appears to be valid to affirm that the SJ strain was more virulent than the LE strain, when used under the experimental conditions previously described. These findings corroborate our early results5.
G roups III, IV, V and VI w ere included in the experim ents aim ing at verifying the effect of reinfections perform ed w ith the same or different strains.
G roup III - This group comprised four mice infected w ith the LE strain and reinfected 90 days later, w ith the same num ber o f cercariae and same strain. They w ere sacrificed at 60 days after reinfection. As far as the microscopic features o f these anim als’ liver are concerned, it seems to be much the same. The most important lesion was represented by granulomatous reaction around eggs spread on the portal tracts and parenchyma. Two different types o f granulomas could be seen clearly: some o f them were small, completely taken by fibrosis, whereas the other ones were large and at the exudative phase. A discrete infiltrate of mononuclear and eosinophil could also be seen in the portal tracts. The hepatocytes showed discrete degenerative phenomena and intense polyploidy. The sinusoids w ere congested and well-dilated, the Kupffer cells w ere hypertrophic and hyperplasic, and sometimes loaded w ith schistosome pigment.
T h e collagen d istrib u tio n in the sm aller granulomas under polarized light was very similar as described in group I. In the larger granulomas - therefore younger - fibrosis detected was less pronounced. The collagen fibers w ere oriented in various directions, w ithout the tendency to form parallel lamellae. These findings were identical to the ones found in the control animals (uninfected) (group VII). It is im portant to note that outside the
granulomas, no significant differences could be seen as far as the am ount o f collagen fibers in relation to the control animals was concerned.
Group IV - W e purpose a further attem pt to compare the importance o f the strain among different reinfections. Thus, the animals o f this group w ere infected with the SJ strain, and reinfected w ith the same strain 90 days later, following the same procedure used for group II. M ice w ere sacrificed at 150 days after primo infection, and the results were compared w ith the ones o f group II. Since two different types o f granulomas (with different ages, some o f them little-sized, fibrous, the other ones larger-sized) could be seen in this group, in general terms the results obtained were sim ilar to the ones from group II. Nevertheless, some differences could be observed: a) the num ber o f granulom as at both evolutive phases (fibrous and exudative granulomas) was larger than in group II; b) as could be seen in group II, the exudative granulomas (causedby reinfection) were predominant in relation to the granulomas in process o f cure by fibrosis (originated from primo infection); c) the peri-portal inflam m atory exudate, that w as com posed o f eosinophils, lymphocytes and plasma cells, was found to be more pronounced than in groups I, II and III; d) the regressive phenom ena in the hepatocytes, such as polyploidy, dilation and sinusoidal congestion w ere m ore severe too, and the amount o f schistosome pigm ent w as larger.
For all the reasons previously shown, w e can conclude that this study demonstrates once more that reinfection w ith the SJ strain is m ore virulent than the one caused by the LE strain.
Groups V and VI - Reinfection w ith crossed
strains was carried out in these groups. A nim als
from group V were infected w ith the LE strain, and reinfected w ith cercariae (SJ strain) at 90 days later; mice from group VI w ere infected w ith the SJ strain, and submitted to reinfection w ith the LE strain. The results obtained were com pared among themselves, as w ell as w ith the ones from groups II and IV.
R a s o P , C o e lh o P M Z , T o p p a N H , M e llo R T . A s p e c ts o f th e g r a n u lo m a to u s r e a c tio n in th e liv e r o f m ic e in fe c te d a n d r e in fe c te d w ith tw o d iffe r e n t g e o g r a p h ic a l s tr a in s «/Schistosom a mansoni. R e v is ta d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 7 : 2 2 1 - 2 2 6 , o u t- d e z , 1 99 4 .
fo llo w in g fundam ental d ifferences could be observed, when groups V and VI were compared: a) the num ber o f granulomas in the animals infected w ith the LE strain and reinfected w ith the SJ strain was found to be larger than in mice infected w ith the SJ strain, and reinfected w ith the LE strain; b) in group V, the am ount o f fibrous granulomas was larger than in group VI; c) the exudative granulomas w ere m ore numerous in group V than in group VI; d) the peri-portal inflammatory exudate was more accentuated in group V; e) the degenerative phenomena in the hepatocytes, as well as polyploidy, w ere m ore evident in the animals from group V.
Finally, group VII (control animals, uninfected) was included in this experiment aiming at verifying a possible interference o f pathologies others than schistosom iasis, specially allowing to compare the behavior o f the periportal connective tissue in infected animals. No hepatic lesions could bedetected in the four animals o f this group.
DISCUSSION
The results o f the present study corroborate our e a r l i e r c o n c lu s io n s 5, s h o w in g a s e v e re r histopathological features as far as the SJ strain of S . m a n s o n i is concerned.
It was p ossible to verify a m ore severe com prom ising o f the hepatocytes (degeneration, necrosis, polyploidy), as well as foci o f larger intralobular inflammatory infiltrate around little foci o f necrosis in the histological preparations related to the SJ strain.
On the other hand, a surprising evidence was the appearance o f a new acute phase in the groups o f reinfected animals, with both heterologous and hom ologous strains. It is im portant to point out that egg-laying connected with the second infection started when prim o infection was in process o f chronic phase o f the disease (about 130 days after prim o infection). In principle, the presence of typical granulomas o f the acute phase in the groups submitted to infection w ith heterologous strain could be explained by some differences found in the constituents o f the soluble egg antigen (SEA), as stated in our earlier study5, which showed a stronger pathogenicity o f the SJ strain, these data being
corroborated by the present w ork. Since these granulomas found to be typical o f the acute phase appeared also in reinfections w ith the same strain o f the parasite, other mechanisms could be present too, altering the control o r modulation o f the granulomatous response, which was maintained in the groups su b m itted to a sin g le in fectio n . M odulation of the inflammatory response around S. m a n s o n i egg at the chronic phase o f the disease was first described by A ndrade & W arren 1. N ow adays, it is m entioned as a w ell-know n phenomenon, as can be seen in B oros’ review2.
Possibly, the factors that could be pointed out as being related to alteration in the process of modulation o f the granulom atous response in reinfections are as follows: overload o f the h ost’s immune system, with m igration o f the worm s from reinfections (skin - lungs - portal system), a larger amount o f antigens deriving either from adult worms related to reinfections or mainly from secretions originated in eggs, that are acknowledged to b e th e fu n d a m e n ta l e le m e n ts in th e immunopathology o f schistosomiasis2 3 H. This a n tig e n ic o v e r lo a d c o u ld s u rp a s s th e immuneregulatory capacity, that was elementary adapted to a lower worm burden.
A larger num ber o f granulom as could also be seen in reinfections w ith the SJ strain. This finding allows the supposition that concomitant immunity connected w ith the SJ strain could be less efficient. Our earlier data showed a weaker immunoprotecti ve capacity in mice with mature infections perform ed with the SJ strain, and submitted to reinfections with homologous and heterologous strains4.
These findings on strengthen o f infection at the chronic phase o f experimental schistosomiasis led us to repeat this experiment w ith a larger num ber of animals to obtain additional corroborative evidence6.
R a s o P , C o e lh o P M Z , T o p p a N H , M e llo R T . A s p e c ts o f th e g r a n u lo m a to u s r e a c tio n in th e liv e r o f m ic e in fe c te d a n d r e in fe c te d w ith tw o d iffe r e n t g e o g r a p h ic a l s tr a in s o/Schistosom a mansoni. R e v is ta d a S o c ie d a d e B r a s ile ir a d e M e d ic in a T r o p ic a l 2 7 :2 2 1 -2 2 6 , o u t- d e z , 1994.
On the other hand, still regarding the murine schistosom iasis, a significant increased level o f interleukin-10 was detected when the Th 1 -response was found to be m odulated7 10 13. Thus, the interleukins 4 and 10, both o f them produced by Th2, should play an im portant role, that is, m o d u la tin g th e in fla m m a to ry re sp o n se s in schistosome infections. The process o f modulation o f the granulom atous response is complex and, b e s id e s th e f a c to r s r e la te d to th e h o s t ’s immunological mechanism, such as cellular response and production o f lym phocyns, other factors connected w ith the parasite must be taken into account, such as: immunodepressor factor derived from S . m a n s o n i (SIDIF) and tetrapeptide TKP (threonine, lysine, proline) produced by clivage of the h ost’s immunoglobulins through proteolytic enzymes o f the parasite that act on the suppression o f m acrophage activation, thus reducing the formation o f granulomas in v i t r o 12. Some factors connected w ith the S . m a n s o n i strain may also act in a different manner, since various S. m a n s o n i strains produce granulom atous reactions with diversified intensity. Our results show that the lesions caused by the SJ strain are always severer, including the ones caused by reinfections. These corroborate our earlier conclusion5.
Further detailed studies are needed to stablish w ith accuracy the mechanism responsible for altering the m odulation o f the granulomatous response in reinfections.
T h erefo re, fu rth e r research dealing w ith quantification o f lymphocynes before and after rein fectio n s, m ainly in terpheron-gam m a and interleukins 2 ,4 ,5 and 10, would be helpful in order to give subtancial agreement related to the factors that are possibly bound to strengthen o f the granulomatous inflammatory response around eggs.
W hen one wants to extrapolate the present results obtained in mice applying them to humans, it is im portant to take into account the enormous phylogenetic distance, as well as the difference in body w eight between these two hosts. Nevertheless, in part, these findings can be helpful to clear up the m atter on the variations o f the anatomoclinic forms o f schistosomiasis mansoni in different geographical regions. These various geographical areas present S . m a n s o n i strains well defined by the different
biological behaviors concerning the various lineages and species o f snails pertaining to the genus B io m p h a la r ia .
RESUM O
N o p r e s e n te e s tu d o , v e r ific o u -s e o c o m p o r ta m e n to h is to p a to ló g ic o d a s in fe c ç õ e s e r e in fe c ç õ e s , h o m ó lo g a s o u h e te r ó lo g a s , d a s c e p a s L E (B elo H o r iz o n te , M G ) e S J (São J o s é d o s C a m p o s , S P ) d e Schistosoma mansoni.
C o n firm o u -se u m a m a io r p a to g e n ic id a d e d a c e p a S J d e
S. mansoni. A s r e in fe c ç õ e s , in d e p e n d e n te m e n te d e te r em s id o f e ita s c o m a m e s m a c e p a (ho m ó lo g a ) o u c e p a d ife r e n te d a p r im o in fe cç ã o (h e te ró lo g a ) d o p a r a s ito , m o s tr a r a m a p r e s e n ç a d e g r a n u lo m a s típ ic o s d a f a s e a g u d a . S ã o d i s c u t i d o s o s p o s s í v e i s m e c a n i s m o s r e s p o n s á v e i s p e l a r e a g u d i z a ç ã o d a r e s p o s t a im u n o p a to ló g ic a n a s r e in fe c çõ e s.
P a l a v r a s - c h a v e s : G r a n u l o m a . R e a ç ã o g r a n u l o m a t o s a . S ch isto so m a m an so n i. C e p a s g e og rá fica s .
ACKNOW LEDGEM ENTS
To CNPQ, FINEP, FA PEM IG , PRPq-U FM G, Brazil and W HO, Switzerland, for financial support. To M rs. Vera de Paula Ribeiro for translating the manuscript. T o M r. A lberto G. dos Santos, M rs. Alice Neni F. Balzuweit and M iss Zenir de Souza for technical assistance.
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