Article available at http://www.parasite-journal.org or http://dx.doi.org/10.1051/parasite/2000071009
LOCAL HEPATIC IMMUNE RESPONSE IN RATS DURING PRIMARY INFECTION WITH FASCIOLA HEPATICA TLIBA O.*, SIBILLE P.*, BOULARD C* & CHAUVIN A.**
Summary:
Résumé
The distribution of lymphocyte subpopulations ( T C D 4 , T C D 8 , +
TCD43
+
+
: RÉPONSE IMMUNITAIRE HÉPATIQUE LOCALE CHEZ LE RAT
INFESTÉ EXPÉRIMENTALEMENT PAR FASCIOLA HEPATICA L'objectif de cette étude était de caractériser
and l g cells), macrophages and eosinophils were +
analysed in the inflammatory infiltrates associated with hepatic
différentes cellules immunitaires
lesions and in hepatic lymph nodes (HLN)
populations
from rats experimentally
lymphocytaires
la distribution
et inflammatoires.
(TCD4+,
TCD8+,
des
Les sous-
TCD43+
cellules lg+), ainsi que les macrophages
8 week post infection ( W P I ) . W e also investigated the fixation of
associés
immunoglobulin isotypes on migrating flukes in the liver. A s early
lymphatiques
as 1 W P I , portal tract areas surrounding migratory tunnels were
expérimentalement
infiltrated with immune and inflammatory cells. The dominant cells
la fixation des différentes
were eosinophils and to lesser extent, macrophages and
douves en migration
et comparé ces résultats avec la
réponse
lymphocytes (TCD4+, T C D 8
humorale systémique.
Dès la première semaine post
infestation
+
and B). Most of the inflammatory
aux lésions
hépatiques
et les
et les
infected with F. hepatica and necropsied 1 , 2 , 3, 4 , 6 and
et dans les
ont été étudiés chez des rats
éosinophiles,
ganglions infestés
par Fasciola hepatica. N o u s avons isotypes d'immunoglobulines
recherché sur les
and immune cells reached the posterior part of flukes, whereas in
(SPI), les espaces portes hépatiques
front of the parasites these cells were fewer in number. Except for
inflammatoires
eosinophils, no immune cells penetrated through granuloma
éosinophiles
et dans une moindre part, des macrophages
consisting of hepatic necrotic cells. A s early as 1 W P I , I g M could
lymphocytes
(TCD4+,
be detected in the liver, and to a lesser extent IgA, l g G
2 a
and
l g G . At 2 W P I , IgE and I g G , began being detected. l g G 2 b
2 c
was
et immunitaires. TCD8+
du parasite,
alors
devant celui-ci. A l'exception
éosinophiles,
follicles in the cortical zone with proliferation of germinal centres
constitué de nombreuses
aucune autre cellule ne pénètre dans le
and medullary cords. The protective role of infiltrating cell
détecte les IgM dans le foie dès la 1SPI, et avec une
cellules hépatiques
populations and immunoglobulin isotypes and possible
intensité les IgA, lgG
mechanisms of immune evasion by the parasite are discussed.
deviennent détectables, et enfin à la 3SPI des ganglions
KEY W O R D S : Fasciola hepatica, rat, lymphocytes, hepatic parenchyma, hepatic lymph node, immunohistochemistry, ADCC.
une prolifération medullaires.
et les lgG . 2b
lymphatiques
zone corticale plusieurs
hépatiques,
follicules
INTRODUCTION asciola hepatica is a liver fluke which causes h u g e e c o n o m i c losses in animal p r o d u c t i o n ( s h e e p and cattle). This parasite can affect a wide range of both domestic and wild species as well as human beings. T h e immune response and resistance to infection or re-infection are quite variable in different F
mécanismes immunitaire
granulome On moindre
les IgE et les
les lgG . 2c
lgG
dans la
microscopiques
et des
1
Au niveau
nous observons
lymphoïdes
des centres germinatifs
qu'elles
nécrotiques.
A 2SPI
avec
cordons
Le rôle protecteur des différentes cellules infiltrées et
les différentes isotypes d'immunoglobulines, MOTS CLÉS : Fasciola hepatica, rat, lymphocytes, parenchyme hépatique, ganglions lymphatiques hépatiques, immunohistochimie, ADCC.
et des
des
detectable at 3 W P I . In H L N , we observed numerous microscopic
2a
cellules
sont des
et B). La plupart de ces cellules
n'atteignent que la partie postérieure sont moins nombreuses
sont infiltrés par des
Les cellules dominantes
d'échappement sont
parasitaires
et les éventuels vis-à-vis du système
discutés.
hosts: sheep, rabbits, mice and goats are very sensitive, whereas rats and cattle can develop partial resistance. After infection, F. hepatica
migrates in its host hepatic-
parenchyma from the first w e e k post-infection
(WPI)
to WPI 8. During this migration period, immune resp o n s e s against the parasite in natural (cattle,
sheep)
or experimental (rat) hosts have b e e n widely studied (Oldham et al.,
1985; Chauvin et ai,
1995; Poitou
al., 1 9 9 3 ) . In rats infected with F. hepatica,
et
Oldham et
al. ( 1 9 8 5 ) observed a proliferative response of spleen * Unité d'Immunopathologie des Maladies Parasitaires. INRA. 37380 Nouzilly, France. ** Unité associée INRA/ENVN Interactions hôte-parasite-milieu, École Nationale Vétérinaire de Nantes, BP 40706, 44307 Nantes Cedex 03, France. Correspondence: Omar Tliba. Tel.: 02 47 42 77 61 - Fax: 02 47 42 77 74 - E-mail:
[email protected]
lymphocytes to the fluke antigens which was detectable during the early phase of the infection. Poitou et al. ( 1 9 9 2 ) described, in infected rats, a similar response o f peripheral blood lymphocytes, splenocytes and thymocytes
to mitogens
such as Concanavalin
P o k e w e e d and to F. hepatica
A and
antigens following infec-
9
tion with F. hepatica. In addition, humoral response and in particular specific immunoglobulin isotypes have b e e n described in a number o f natural or e x p e rimental hosts such as Rat (Poitou et al., 1 9 9 2 ) , Man (Pailler et al, 1 9 9 0 ) and S h e e p (Chauvin et al, 1 9 9 5 ) . T h e local i m m u n e response in the area surrounding young flukes during migration through the liver which may play a role in resistance m e c h a n i s m s has b e e n e x p l o r e d less. Chauvin et al, ( 1 9 9 6 ) s h o w e d that the migratory tunnels produced by juvenile flukes in sheep liver a p p e a r e d as focal areas o f necrosis surrounded by infiltrating inflammatory cells, in particular numerous m a c r o p h a g e s , eosinophils and O v C D 4 lymphocytes. Moreover, during chronic infection and re-infection in s h e e p , the n u m b e r o f C D 8 l y m p h o c y t e s b e c a m e greater than that of C D 4 ( M e e u s e n et al, 1995). However, local immune cellular and humoral responses have not b e e n studied in hepatic lesions and hepatic lymph n o d e s (HLN) o f less susceptible hosts such as cattle or rats. +
+
+
T h e aim o f the present study was to describe, in rats, the localisation a n d kinetics o f cellular r e s p o n s e s ( m a c r o p h a g e s , e o s i n o p h i l s , lymphocyte s u b p o p u l a tions) and humoral responses (IgM, IgG subclasses, IgA and IgE) b e t w e e n WPI 1 and WPI 8 in the areas surrounding juvenile flukes and in the surrounding hepatic parenchyma. T h e i m m u n e response in HLN w a s also explored.
MATERIALS AND METHODS EXPERIMENTAL DESIGN
W
istar male rats (Iffa Credo, Arbresle, F r a n c e ) approximately 13 w e e k s old were used. In this protocol, eight groups o f two rats which w e r e infected orally with 50 metacercariae, and o n e group of 12 rats (uninfected animals) w e r e killed at regular intervals after infection. G r o u p G l , G2, G 3 , G4, G 6 and G 8 w e r e killed at 1, 2, 3, 4, 6 and 8 WPI, respectively. T w o animals in the control group w e r e n e c r o p s i e d at 0, 1, 2, 3, 4, 6 and 8 WPI. D o s e effect w a s studied: G 1 . 1 , G 1 . 2 and G 1 . 3 w e r e infected with 50, 100 and 2 0 0 metacercariae respectively. NECROPSY AND HISTOLOGICAL PREPARATION
Rats w e r e killed by chloroform inhalation. T h e livers and HLN w e r e immediately removed. Several 1 c m s a m p l e s o f liver or HLN w e r e dissected from the macroscopically visible lesions in e a c h rat or from the control rat, e m b e d d e d in O C T c o m p o u n d (Tissue-tek; Miles, USA) and rapidly immersed in isopentane cooled with dry ice. All samples w e r e stored at - 8 0 ° C until further processing. For e a c h animal, 7-8 µm thick sec3
10
tions o f six to ten liver samples and o n e HLN sample w e r e serially sectioned using a cryostat (Rua, F r a n c e ) , and air-dried overnight. STAINING E o s i n o p h i l s a n d m a c r o p h a g e s w e r e identified o n hemalun-eosin (HE) or May-Grünwald Giemsa ( M G G ) stained sections. Lymphocyte subpopulations and different immunoglobulin subclasses w e r e characterised by immunohistochemistry with IgG1 m o u s e m o n o clonal antibodies (Mabs). All primary Mabs w e r e purc h a s e d from Serotec (Argène Varilhes, F r a n c e ) . Pan-T lymphocytes ( C D 4 3 cells), T C D 4 lymphocytes, T C D 8 lymphocytes and m a c r o p h a g e s w e r e identified using Mab W3/13 (1:50), Mab W3/25 (1:75), Mab O X - 8 ( 1 : 3 0 ) and Mab ED2 ( 1 : 8 0 0 ) respectively. For the isotypes o f immunoglobulins IgM, IgE, IgA, I g G , I g G , I g G and I g G w e used, respectively, Mab MARM-4 ( 1 : 3 0 0 0 ) , M a b MARE-1 ( 1 : 4 0 0 ) , M a b MARA-1 ( 1 : 4 0 0 ) , M a b MARGE 1-2 ( 1 : 8 0 0 ) , Mab MARGE 2-A61 ( 1 : 8 0 0 ) , Mab MARG 2b-8 ( 1 : 8 0 0 ) , and Mab MARGE 2c5 ( 1 : 4 0 0 ) . For immunohistochemical staining, w e used the technique previously described by Cordell et al, ( 1 9 8 4 ) , modified by Pepin et al, ( 1 9 9 2 ) . Tissue sections were fixed in a c e t o n e at - 20° C for 10 min, air dried for 20 min and rehydrated for 15 min with Tris-buffered saline (TBS, 0.05 M Tris, 0.15 M NaCl, pH 7.6). S e c tions w e r e incubated with MAb for 30 min in a humid c h a m b e r . After three w a s h e s in TBS, sections w e r e covered with a 1:100 dilution o f rabbit anti-mouse Igs (depleted o n rat Igs) ( D a k o , D e n m a r k ) for 30 min in a humid c h a m b e r . After washing three times in TBS, they w e r e incubated with a 1:100 dilution o f APAAP c o m p l e x (soluble c o m p l e x e s o f calf intestinal Alkaline P h o s p h a t a s e a n d M o u s e m o n o c l o n a l Anti-Alkaline Phosphatase ( D a k o ) ) for 30 min in a humid c h a m b e r . After washing twice in T B S and twice in Tris HC1 0.1 M pH 8.2, the slides w e r e stained with the filtered substrate: 20 mg Naphtol AS-TR p h o s p h a t e (Sigma, SaintLouis, USA), 2 ml dimethylformamide ( P r o l a b o ) , 30 µg levamisole (Sigma), 100 ml Tris-HCl 0.1 M pH 8.2 and 100 mg Fast Red TR salt (Sigma) for 20 min. Slides w e r e w a s h e d in water for 10 min, counterstained with hematoxylin (Sigma) for 30 to 60 s and mounted in glycerol gelatine. +
+
+
1
2a
2b
2 c
RESULTS HEPATIC PARENCHYMA Lesion description
A
t necropsy, there w e r e n o appreciable lesions in the livers o f control animals, neither m a c r o -
scopically nor microscopically. In infected ani-
mals, n u m e r o u s macroscopic, tortuous migratory path-
ways b e g a n to b e o b s e r v e d after the s e c o n d w e e k of infection. In groups G 6 and G8, the livers presented irregular surfaces, with nodular and pale areas. T h e main bile duct appeared to b e enlarged, wide and with flukes inside. Microscopically, six juvenile flukes w e r e o b s e r v e d in the hepatic parenchyma (in t w o animals at 1 WPI, in o n e animal at 2 WPI, in o n e animal at 3 WPI and in t w o animals at 4 WPI (Fig. 1a)). T h e most striking feature o f the hepatic parenchyma c o n c e r n e d the n u m e r o u s migratory tunnels f o r m e d b y j u v e n i l e f l u k e s (Figs, lb and lc). Eosinophils invaded the center o f these pathways (Fig. 1: arrow). T h e y w e r e the p r e d o minant cell type a m o n g the m o n o n u c l e a r cells, macrop h a g e s and neutrophils w h i c h accumulated near the necrotic hepatocytes. At 3 W P I w e observed, likewise, pericholangitis and the beginning o f periportal fibrosis (Fig. Id). B a n d s of collagen of variable size linked the migratory tunnels to adjacent structures such as portal triads and hepatic veins. T h e y subdivided the paren-
chyma into a series of regular lobules. At 6 WPI, hyperplastic ductular epithelia associated with proliferating portal areas (Fig 1d) w e r e observed. At 8 WPI, eosinophils a n d o t h e r inflammatory and immune cells spread into the surrounding parenchyma. W e also o b s e r v e d the formation o f b a t c h e s o f bile ductules (Fig. Id). T h e size o f these lesions increased during the infection and resulted in cirrhosis formation. Local cellular response In control animals, immunohistological staining showed that T lymphocytes and m a c r o p h a g e s w e r e distributed h o m o g e n e o u s l y throughout the hepatic parenchyma (Figs. 2a, 2c, 2e). In infected animals, microscopic examination s h o w e d that, as early as 1 WPI, the dominant cells w e r e eosinophils (Fig. lb). At this moment, immune and inflammatory cells and particularly T lymphocytes w e r e found in small numbers in the area surrounding the parasite (Fig. 2f). T h e r e w e r e f e w e r m a c r o p h a g e s and lymphocytes than eosinophils, and
Fig. 1. - Haematoxylin/eosine stained frozen section showing: (a) Liver flukes at 4 WPI (x 200); (b) Granuloma at 1 WPI (x 200); (c) Granuloma at 4 WPI (x 200); (d) Hyperplastic ductular epithelium and bile ductules formation at 6 WPI (x 400). Gr: granuloma; PC: pericholangitis; PF: periportal fibrosis; Arrow: eosinophils.
11
Fig. 2. - Immunohistology staining of frozen sections in hepatic parenchyma showing: (a) TCD8* in uninfected control animal (x 200), (b) TCD8 at 2 WPI (x 200); (c) macrophage in uninfected control animal (x 100), (d) macrophage at 2 WPI (x 100); (e) TCD43 in uninfected control animal (x 200), (f) TCD43 at 1 WPI (x 400); (g) IgM in uninfected control animal (x 200), (h) IgM at 1 WPI (x 200). PS: portal space; F.h: Fasciola h e p a t i c a ; V: centrolobular vein. +
+
12
+
their n u m b e r increased clearly at 2 WPI (Figs. 2d and 2 b ) . As o f 2 or 3 WPI, w e noted an inflammatory infiltrate surrounding the juvenile flukes (Fig. 2b) which is w a s greater in the posterior part o f the flukes. T h e granuloma was surrounded b y an area of infiltrating inflammatory cells, particularly m a c r o p h a g e s , lymphocytes ( C D 4 , C D 8 , C D 4 3 ) , eosinophils and a few neutrophils. W e also found these cells in fibrosis. T h e structure o f these granulomatous lesions w a s similar in all infected rats but their size increased from WPI 1 to WPI 8 ( T a b l e I). S o m e B lymphocytes ( I g M cells) w e r e observed in the inflammatory infiltrate in the area surrounding the juvenile flukes (Fig. 2h) and in the migratory tunnels. W e also n o t e d a decrease in the n u m b e r o f these cells in groups killed at 4, 6 and 8 WPI. Increasing n u m b e r s o f parasites resulted in an increase in the recruited i m m u n e and inflammatory cells ( g r o u p G 1 . 1 - G 1 . 3 ) e x c e p t for B cells w h i c h remained at a constant frequency. +
+
+
+
the tegument. W e also noted that most o f the IgE antib o d i e s w e r e b o u n d to eosinophils (Fig. 3b). HEPATIC LYMPH NODES T h e HLN o f infected animals e x h i b i t e d n u m e r o u s microscopic follicles in the cortical z o n e with proliferation of germinal centres and medullary cords (Figs. 4a, 4b, 4c, 4d). Hypertrophy o f HLN was clearly observed as o f 3 WPI. Immunohistological staining s h o w e d that infiltration o f immune and inflammatory cells in HLN and in hepatic parenchyma w e r e related, the staining intensity increasing from 1 to 2 WPI, then being maintained throughout the infection. T h e majority o f T lymphocytes ( C D 4 3 , C D 4 and C D 8 ) w e r e located in follicles (Figs. 4 c , 4d) and in paracortical areas whereas moderate numbers o f these cell subsets were located in the connective tissues o f the medulla. Macrophages ( E D 2 cells) were located especially in the follicle zone but also in the paracortical area and the medulla (not s h o w n ) . B-lymphocytes (IgM cells) w e r e observed in lymphoid follicle centres (Fig. 4b). T h e plasma cells were found on the border o f the germinal centres and in large numbers in the medullary cords and the medulla. Numerous eosinophils w e r e also present in the medulla but to a lesser extent than in the hepatic parenchyma. Staining o f all immunoglobulin isotypes was generally greater at 2 WPI and remained throughout the infection, except for I g G which decreased at 6 WPI. +
+
+
+
+
Cells Group Group Group Group Group Group Group
TCD43* C Gl G2 G3 G4 G6 G8
TCD4*
TCD8*
Macrophage
(IgM cells) +
+
+
+
+
+
++ +++ +++ +++ +++ +++
++
++ +++ +++
++ +++ +++ +++ +++ +++
++ +++ +++ + + +
+++ +++ +++ +++ +++
+++
+++ +++
2c
Table I. - Cellular immune response in hepatic parenchyma. Number of stained cells per microscope areas was evaluated as follows: - : absence of staining; +: faint staining; ++ : medium staining; +++: intense staining).
Local humoral r e s p o n s e As early as 1 WPI, IgM could b e detected in the liver (Figs. 2b), and to a lesser extent IgA, I g G and I g G . At 2WPI, IgE (Fig. 3b) and IgG, b e g a n being detected. I g G appeared at 3 WPI. T h e amount o f detected antib o d i e s ( A b ) w a s directly correlated with the fluke burden. 2 a
2 b
2 c
All immunoglobulin isotypes exhibited a similar distribution. T h e staining intensity increased from 1 WPI to 8 WPI for IgM, IgA and IgG, w h e r e a s I g G , I g G a n d IgE h a d d e c r e a s e d at 8 W P I . I g G started to decrease at 6WPI. T h e plasma cells w e r e found in the c o n n e c t i v e tissue or in the d a m a g e d parts o f the liver. T h e y w e r e o b s e r v e d in the inflammatory infiltrate, in the granuloma lesions, in the area surrounding the parasites (Figs. 2b, 3d and 3b), in the portal tract and in the fibrotic strands (Fig. 3f). T h e tegument o f juvenile flukes was covered b y immunoglobulins o f all isotypes e x c e p t for IgE (Figs. 2h, 3d, 3h). T h e parasite c a e c a w e r e also labelled but with less intensity than 2a
2 c
2 b
DISCUSSION
T
he hyperplasia o f lary cords o f the tion of leukocyte lesions indicated strong the parasite.
lymphoid follicles and HLN and the intense and immune cells in local immune reaction
medulinfiltrahepatic against
T h e cellular r e s p o n s e was characterised by extensive inflammatory infiltrate in hepatic lesions in the areas surrounding the parasites and migratory tunnels, and took place at 2 WPI. This infiltrate contained T and B lymphocytes, m a c r o p h a g e s and numerous e o s i n o phils. During the first w e e k , the infiltrate was not o b e r v e d to b e in contact with the juvenile flukes, suggesting that, early on in the infection, cellular responses did not reach the parasite. This e n a b l e d the parasite to migrate unhindered through the liver tissue. At 2 WPI, most of the immune cells w e r e detected in the hinder part o f flukes, w h e r e a s there w e r e fewer o f these cells in front o f the parasite, w h i c h is consistent with other studies: in s h e e p liver, Chauvin et al., (1996) s h o w e d juvenile flukes migrating into healthy tissue. This suggests that liver flukes e s c a p e from the immune resp o n s e w h i c h is d e v e l o p e d b e h i n d them.
13
Fig. 3- - Immunohistology of frozen sections in hepatic parenchyma showing: (a) IgE in uninfected control animal (x 100) and (b) at 2 WPI (x 200); (c) IgA in uninfected control animal (x 400) and (d) at 4 WPI (x 200); (e) IgG, in uninfected control animal (x 100) and ( 0 at 8 WPI (x 200); (g) I g G in uninfected control animal (x 200) and (h) at 2 WPI (x 100). 2a
14
Fig. 4. - Immunohistology of frozen sections in hepatic lymph node: (a) IgA at 2 WPI (x 100); (b) IgM at 2 WPI (x 100); (c) CD8 at 2 WPI (x 100); (d) CD43 at 2 WPI (x 100). F: follicle; M: medulla. +
+
Granuloma structure could b e o b s e r v e d in the migration pathway behind the flukes. This local immune resp o n s e was characterised by the infiltration o f only eosinophils into the granuloma and also around the flukes. Conversely, an increase in T cells, B cells, macrophages and eosinophils was observed in the areas surrounding these migratory tunnels. This cell distribution w a s similar to the description given b y Chauvin et al, (1996) in s h e e p liver during experimental infection with F. hepatica. B a e z a et al, (1994) noted an early reduced systemic inflammatory r e s p o n s e during the first t w o w e e k s in rats infected with F. hepatica, suggesting impairment o f the host d e f e n c e m e c h a n i s m s by the flukes. Moreover, F. hepatica may avoid inflammatory responses by means o f rapid migration through healthy p a r e n c h y m a (Chauvin et al, 1996). T h e formation o f p r o n o u n c e d peri-lobular fibrosis and the p r e s e n c e o f large numbers o f C D 8 cells in the fibrotic strands after 8 WPI w e r e features of chronic infection in r e s p o n s e to continuous stimulation with antigen. This was confirmed b y the persistence of lymphoid follicles in the HLN at 8 WPI. Masake et al, +
( 1 9 7 8 ) s h o w e d , in m i c e experimentally infected with F. hepatica, that the regeneration of damaged liver cells w a s c o m p l e t e d t w o months after infection, although hyperplasia persisted in bile ducts containing flukes. A similar observation w a s n o t e d in our study; at 8 WPI, e x c e p t for the lesion areas which turned to cirrhosis, the livers l o o k e d healthy, in contrast with the totally d a m a g e d livers o b s e r v e d at 4 WPI. T h e local cellular r e s p o n s e a p p e a r e d to b e d o s e dependent. Inflammation intensity increased with parasite burden, indicating that the local immune response w a s d e p e n d e n t o n f l u k e n u m b e r . This r e s p o n s e increased from 1 WPI to 8 WPI, which may have b e e n due to increasing amounts of released excretion-secretion (ES) antigens and the n u m b e r and size o f lesions in the liver. Local i m m u n e responses may b e correlated with systemic immune responses. Poitou et al, (1993) observed a significant increase in numbers o f B lymphocytes in the spleen o f experimentally infected rats from 3 WPI to 7 WPI. This w a s correlated with i) the development o f lymphoid follicles, particularly in HLN early after
15
infection (1 W P I ) , ii) marked mobilisation o f B cells in hepatic parenchyma (1 W P I ) , and iii) high production o f various different Ab isotypes o b s e r v e d in the liver and serum. In s h e e p , the observations of Chauvin et al. ( 1 9 9 6 ) w e r e similar. In addition, Poitou et al., ( 1 9 9 3 ) s h o w e d systemic eosinophilia w h i c h correlated with the early and intense local eosinophilic infiltration also o b s e r v e d in this study. This e o s i n o p h i l recruitment could b e caused by eosinophil chemotactic factors (ECF) detected in F. hepatica ES products (Horii et al, 1 9 8 6 ) . During experimental rat fasciolasis, the percentage o f circulating C D 4 and C D 8 T lymphocytes d e c r e a s e d from 1 W P I to 8 WPI (Poitou et al, 1 9 9 3 ) . During this p e r i o d , in liver p a r e n c h y m a , C D 4 a n d C D 8 cell recruitment increased in the area surrounding the fluke migration pathways and this could explain w h y their numbers d e c r e a s e d in the b l o o d . In s h e e p , Moreau ( 1 9 9 7 ) also s h o w e d that circulating C D 4 and C D 8 T lymphocytes d e c r e a s e d during fasciolasis b e t w e e n 3 and 6 WPI. However, in s h e e p liver, Chauvin et al. ( 1 9 9 6 ) o b s e r v e d an increase in the n u m b e r of C D 4 T - l y m p h o c y t e s , and n o increase in the n u m b e r o f C D 8 T-lymphocytes. This difference observed in C D 8 T-lymphocyte recruitment in hepatic parenchyma betw e e n rat (resistant host) and s h e e p (sensitive host) indicates that these cells may play a role in resistance to the parasite. +
+
+
+
+
+
+
+
+
Among the most important features of the local humoral responses, w e o b s e r v e d a high level o f IgM, w h i c h correlates with systemic humoral r e s p o n s e previously described in the rat (Poitou et al, 1993; Wedrychowicz et al, 1 9 8 7 ) and in s h e e p fasciolosis (Chauvin et al., 1 9 9 5 ) . IgM antibodies are an indicator of a primary r e s p o n s e against an antigen (Takahashi et al, 1990). T h e persistence, during the entire course of infection, o f a high level o f IgM covering the fluke tegument might b e e x p l a i n e d b y the sequential secretion o f different parasite antigens (Poitou et al, 1 9 9 2 ) , b y a rapid turnover o f the outer glycocalyx of the flukes ( H a n n a et al, 1980a; Duffus et al, 1 9 8 0 ) , or by a high level o f B cells stimulation b y carbohydrate epitopes. Glauert et al., ( 1 9 8 5 ) suggest that, as eosinophils do not express Fcµ receptor (McEwen et al., 1992), this IgM deposition o n fluke tegument may inhibit eosinophil a c c e s s to the parasite. T h e s a m e IgM responses w e r e n o t e d in other hosts such as s h e e p (Chauvin et al., 1 9 9 6 ) and to other trematodes such as Schistosoma mansoni (Khalif et al, 1 9 8 5 ) . Thus IgM may act as a blocking isotype during fasciolasis. Another b l o c k i n g isotype o b s e r v e d in this study is I g G . This antibody has b e e n described as a blocking factor in immunity to schistosome and in particular in I g G - m e d i a t e d eosinophil cytotoxicity (Khalif et al, 1 9 8 5 ) . T h e authors suggested that the existence o f a
c o m m o n receptor shared b e t w e e n I g G and I g G o n the eosinophil surface could b e the c a u s e o f this b l o c king effect. In this study, I g G antibody w a s also detected at the third W P I in hepatic p a r e n c h y m a and in HLN, suggesting that F. hepatica may e n h a n c e ineffective humoral responses b y two different m e c h a nisms: o n e involving IgM w h i c h coat the parasite and avoid ADCC (Antibody dependent cellular cytotoxicity), the s e c o n d involving I g G , inducing competitive b l o c king of I g G receptors o n the eosinophil surface. In addition, these antibodies are k n o w n to react with T-cell independent antigens ( D e r Balian et al., 1 9 8 0 ) such as carbohydrate m o l e c u l e s . T h e s e glycoproteins also induce a polyclonal n o n specific B cell r e s p o n s e w h i c h blurs the specific i m m u n e r e s p o n s e . 2 c
2 a
2 c
2c
2 a
I g G and IgE are considered to b e anaphylactic antib o d i e s in the rat (Capron et al, 1 9 7 7 ) . T h e s e antibodies are k n o w n to b e involved in c o o p e r a t i o n with phagocytic cells such as m a c r o p h a g e s or eosinophils in ADCC. I g G m o n o c l o n a l antibodies directed against schistosomula have b e e n s h o w n to confer highly significant levels o f protection against Schistosoma mansoni infection (Capron et al, 1 9 8 3 ) . IgE has also b e e n demonstrated to play a pre-eminent role in eosinophil cytotoxicity against parasitic diseases such as rat schistosomiasis (Capron et al., 1 9 8 1 ) . In the present work, IgG antibodies w e r e detected in the liver parenc h y m a and in HLN as early as 1 WPI. IgE antibodies a p p e a r e d in the hepatic p a r e n c h y m a at 2 WPI, suggesting that F. hepatica also i n d u c e s an effective humoral r e s p o n s e b y producing two effector antibodies, I g G and IgE, w h i c h may play a protective role in fasciolosis d e p e n d i n g o n the b a l a n c e b e t w e e n protective and antagonist antibodies. 2 a
2 a
2 a
2 a
A m o n g other antibodies w h i c h may play a protective role in disease, IgG, antibodies are involved in effector m e c h a n i s m s o f acquired resistance and schistosome killing (Capron et al., 1 9 7 7 ) , I g G antibodies have lethal activity towards schistosoma in vitro in the pres e n c e o f c o m p l e m e n t (Fatima et al., 1 9 8 4 ) , and IgA antibodies are involved in mechanisms implying eosinophils conferring protection against schistosoma (Grezel et al., 1993). Van Milligen et al., ( 1 9 9 8 ) , demonstrated that, u p o n challenge o f i m m u n e rats with F. hepatica in an ex vivo gut segment, n e w e x c y s t e d juvenile flukes that migrated through the m u c o s a w e r e coated with I g G , and I g G antibodies and surrounded b y eosinophils, w h i c h correlated with the e x p r e s s i o n o f protection, suggesting that juvenile flukes are killed by eosinophil-mediated cytotoxic r e s p o n s e involving IgG antibodies. In rat liver, the role o f these antibodies has not b e e n explored. 2 b
2 a
2 c
2a
16
In rat fasciolosis, further studies are n e e d e d to e x p l o r e the role o f the parasite in regulating the host i m m u n e response. Particularly, different cytokines secreted b y
lymphocytes and other i m m u n e cells in b l o o d , hepaticp a r e n c h y m a and HLN s e e m to b e important in determining parasite rejection or m a i n t e n a n c e . T h e role o f in the differentiation o f T
F. hepatica
lymphocytes
should b e further investigated.
Clinical 440.
and
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Reçu le 2 3 février 1 9 9 9 Accepté le 8 novembre 1 9 9 9
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