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Echinostoma caproni in mice: Ultrastructural studies on the formation of immune complexes on the surface of an intestinal trematode
lnrernurronol Journalfor Primed in Great Britain
Parasiloiogy
Vol. 20, No. 7. pp. 93S941.
1990 0
ECHINOSTOMA
CAPRONI
co2tL7519/90 $3.00 + 0.00 Pergamon Press p/c Socieryfor Pnrosiro/ogy
IN MICE: ULTRASTRUCTURAL
ON THE FORMATION OF IMMUNE COMPLEXES OF AN INTESTINAL TREMATODE PAUL E. SIMONSEN,*~
1990 Awmlim
B. JYDING
VENNERVALD*
STUDIES ON THE SURFACE
and A. BIRCH-ANDERSEN~
* Danish Bilharziasis Laboratory, Jaegersborg Alle 1 D, 2920 Charlottenlund, Denmark fDepartment of Biophysics, Statens Serum Institut, Amager Boulevard 80,230O Copenhagen S, Denmark (Received 23 March 1990; accepted 11 June 1990) AbStrzW-SIMONSEN P. E.,~ENNERVALD B.J. and BIRCH-ANDERSEN A. 1990.Echinostoma caproni in mice: ultrastructural studies on the formation of immune complexes on the surface of an intestinal trematode. International Journalfor Parasitology 20: 935-941. The binding of mouse antibodies to the surface antigens of juvenile and 7 and 28 day old Echinostoma caproni was examined by transmission electron microscopy of thin sections of parasites, which were treated with antibodies in a double sandwich technique with ferritinconjugated antibody. The surface of freshly recovered mature adult parasites was covered with an irregular but often rather intensive mouse antibody containing matrix, which probably represents a layer of mouse antibody/parasite antigen complexes. The complexes were lost after in vitro culturing of the parasites for 24 h, but incubation of the in vitro-maintained antibody-negative adult parasites with immune mouse serum led to reformation of a similar but less intensive cover with immune complexes. Juvenile and young stages of E. caproni, which had never been exposed to host antibodies, obtained a layer of immune complexes on their surface after incubation with immune mouse serum in vitro. In both young and mature parasites, the antibody-antigen complexes were observed to be rather loosely attached to the outer surface of the parasites, where the antigens probably constitute a part of the irregular glycocalyx of the organisms. It may also be that the antigens are present as isolated excretions along the surface of the parasites. Several sections indicated that the parasite surface antigens may be present in the tegument in vesicles which fuse with the outer membrane of the parasite whereby their contents are released to the exterior. INDEX KEY WORDS: Echinostoma caproni; intestinal trematode; mice; antibodies; immune complexes; ultrastructure; ferritin-conjugated antibody labelling; surface antigens; antigen shedding; tegumental
Infections with E. cuproni in mice induce a significant antibody response to the surface of the parasites (Simonsen & Andersen, 1986). The antibodies can be measured both in serum and on the surface of the parasites in the mouse intestine. Antibodies to the surface of juvenile parasites appear in serum at the same time as resistance to reinfection develops. However, antibodies which are bound to the live parasite surface are rapidly lost in vitro, in a process involving shedding of antigens from the parasites at a high rate (Simonsen & Andersen, 1986; Andresen, Simonsen, Andersen & Birch-Andersen, 1989). The effect of the antibodies on the E. caproni surface, and their significance in regulating the parasite population in the host, are presently not known. In the present investigation, the interaction between antigens on the surface of E. caproni and mouse antibodies was examined at the ultrastructural level, using a ferritin-conjugated antibody technique. The purpose was to localize surface antigens, and to visualize the dynamics of immune complex formation on the surface of E. caproni.
INTRODUCTION
MICE infected with Echinostoma caproni constitute a useful laboratory model for studies on intestinal trematodes in the final host (Christensen, Odaibo & Simonsen, 1988). Detailed observations have been documented on the parasite population regulation in this model (Christensen, Knudsen & Andreassen, 1986; Odaibo, Christensen & Ukoli, 1988, 1989). The cause of a primary E. caproni infection in mice is infection dose dependent. Light infections with six parasites are expelled in 68 weeks, whereas heavy infections with 25 parasites are not expelled. However, approximately 2 weeks after a primary infection of any size, complete resistance to reinfection develops, which seems to be of an immunological nature (Christensen et al., 1986). Resistance to reinfection does not affect the initial infection and therefore this host-parasite relationship represents an example of concomitant immunity.
t To whom all correspondence
should be addressed. 935
936
P. E. SIMONSEN, B. J. VENNERVALD and A. BIRCH-ANDERSEN MATERIALS
AND METHODS
Mice and parasites. Female
NMRI mice were obtained from Bomholtgaard Ltd, Denmark. Only mice more than 6 weeks old at the start of the experiments were used in these studies. Echinostoma caproni is identical to the population also named E. revolutum in previous work from the Danish Bilharziasis Laboratory (see Christensen et al., 1988). The life cycle is maintained in mice and Biomphalariu glubrata snails. Mice are infected by stomach tube feeding with metacercariae obtained by dissection of infected snails. Sera. Mouse blood samples were obtained by cardiac puncture under deep ether anaesthesia. Normal non-immune serum was obtained from uninfected mice, whereas immune serum was from a group of mice infected with 25 metacercariae 4 weeks previously. Preparation of juvenile parasites. Juvenile parasites were produced by in vitro excystation of metacercariae with trypsin and sodium taurocholate as previously described (Simonsen & Andersen, 1986). After excystation they were washed in PBS, pH 7.2, and prefixed in 2% formalin in PBS. Before serum incubations they were washed three times in PBS, once in 50 mM-NH,Cl and three times in PBS to block any reactive aldehyde groups. They were then divided into two groups which were incubated for 30 min with normal or immune mouse serum (1:20 in PBS), washed twice with PBS, incubated for 30 min with rabbit-anti-mouse antibody (1:40; Dakopatt), washed twice with PBS, incubated for 30 min with ferritin-conjugated goat-anti-rabbit IgG (1:25; MilesYeda Ltd, Israel) and finallv washed three times with PBS. Another batch ofjuvenile parasites was washed with PBS for removal of the excystation medium and then divided into two groups for incubation with immune or control serum in the living state, before they were killed with 3% glutaraldehyde in cacodylate buffer (pH 7.2) at room temperature for 30 min. No difference was noted between the preservation of ultrastructure or antigenicity of the organisms from the first and second batch, and consequently the results are not kept separate in the following experiments. Preparation of 7 day old parasites. The parasites were recovered from mice 7 days after infection with 25 metacercariae. After washing in PBS, they were incubated in normal or immune serum for 30 min, followed by washing three times in PBS. Further incubation in rabbit-anti-mouse antibody and ferritin-conjugated goat-anti-rabbit IgG was carried out as described for juvenile parasites. Finally, the parasites were killed by incubation with 3% glutaraldehyde in cacodylate buffer (pH 7.2) at room temperature for 30 min, before washing three times with PBS. Preparation of mature adult parasites. Mature adult parasites were obtained by dissection of mice 28 days after infection with 25 metacercariae. The parasites were washed rapidly in PBS. Adults for direct examination were then prefixed immediately with 2% formalin in PBS, washed three times in PBS, once in 50 mM-NH&l and three times in PBS. The parasites were then treated with rabbit-anti-mouse
antibody and ferritin-conjugated goat-anti-rabbit IgG, and washed in PBS as described for juvenile parasites. Other adults were maintained in vitro in RPM1 1640 tissue culture medium supplemented with 25 mM-HEPES, 22 mM-NaHCO, and 50 pg-mll ’ gentamycin (Garamycin, Schering Corp., U.S.A.) for 24 h at 37°C before prefixation with 2% fonnalin in PBS. After washing in PBS and 50 mM-NH,CI as above, these adults were incubated in normal or immune mouse serum (1:10 in PBS), rabbit-anti-mouse antibody and ferritin-conjugated goat-anti-rabbit IgG, before washing in PBS as described for iuvenile parasites. After the above preparations, all adult parasites were cut transversely while still soaked in PBS, and only the anterior third of the worms were further prepared for electron microscopy. Fixation and embedding for transmission electron microscopy. After the treatments described above, whole juvenile and 7 day old parasites, and the anterior third of adult parasites were transferred from the PBS to 45°C melted 1.5% Noble agar (Difco) in cacodvlate buffer pH 7.2. After cooling, small-blocks of worms embedded in the agar were fixed in 3% nlutaraldehvde in 0.1 M-cacodvlate buffer DH 7.2 at 4°C overnight. Furiher preparation for sectioning and electron microscopy followed the procedure previously described (Andresen ef al., 1989). RESULTS
adult parasites Freshly recovered 28 day old E. caproni, fixed immediately after recovery and incubated with ferritin-labelled conjugate, were examined in the transmission electron microscope. The parasites were covered with an irregular but often rather intensive matrix of material on the outer tegumental membrane (Fig. la). The presence and position of ferritin molecules in the matrix, together with the fact that it has previously been demonstrated that antigens are continuously excreted from the surface of live E. caproni (see Andresen et al., 1989), suggest that the matrix was made of complexes of parasite antigen and mouse antibody. The membrane itself was never labelled with ferritin, however, implying that parasite surface antigens are not integrated into the membrane, but rather seem to be intermingled with an irregular glycocalyx or excreted to the exterior from the surface of the parasites. Maintenance of the mature adult parasites for 24 h in vitro resulted in loss of the covering matrix from the surface of the parasites, and ferritin labelling was no longer present (Fig. lb). Only a thin regular layer of glycocalyx was observed on the outside of the tegumental membrane. The immune complexes which were present qn freshly recovered parasites had Mature
FIG. 1. Transmission electron micrographs of the surface of 28 day old E. caproni, after treatment with murine sera and ferritinconjugated antibody. F, ferritin molecules; IC, matrix of immune complexes; M, outer tegumental membrane; V, membranebound vesicle. Scale bars = 400 nm. (a) Freshly recovered parasite. The arrow indicates a vesicle which is fused with the outer tegumental membrane. (b) Parasite after 24 h of in vitro maintenance followed by incubation in normal mouse serum. Note absence of immune complexes. (c)Parasite after 24 h of in vitro culture followed by incubation in immune mouse serum. Note presence of immune complexes. The arrow indicates a vesicle which is fused with the outer tegumental membrane, thus establishing direct contact with the exterior.
Immune complexes on E. cuproni surface
937
938
P. E. SIMONSEN,B. J. VENNERVALDand A. BIRCH-ANDERSEN
Immune complexes on E. cuproni surface apparently been sloughed off during the in vitro culture. In vitro exposure of the cultured antibody-negative adult parasites to immune mouse serum led to reformation of immune complexes on the outside of the parasites (Fig. lc). Although large amounts of antigen/antibody complexes were present on the parasite surface, the aggregates were usually smaller and the covering less intensive than seen with freshly recovered adult parasites. The re-formation of immune complexes after shedding of the original complexes indicates that antigenic material must be continuously released from the tegument to the parasite surface and further to the exterior. The tegument of all 28 day old E. cuproni was densely packed with membrane-bound vesicles, especially near the outer tegumental membrane (Fig. la-c), indicating an active secreting surface. The morphology of the vesicles was rather variable, but most of them belonged to the circular or elongate types described previously (Andresen et al., 1989). Occasionally vesicles were seen to fuse with the outer tegumental membrane (Fig. la and c), and thereby releasing their contents to the exterior. At least part of the released material was antigenic, as indicated by the ferritin labelling. Younger stages Seven day old E. cuproni, freshly recovered from mouse intestines, exhibited no immune complexes on their surface, when examined by the ferritin labelling technique (Fig. 2a). This result is not surprising, since antibodies against the parasite surface are not produced by mice 7 days after a primary infection (Simonsen & Andersen, 1986). Some cloudy loose material was observed exterior to the outer leaflet of the tegumental membrane, which may have been uncomplexed antigens. Incubation of the parasites in immune mouse serum led to the formation of an irregular but intensive layer of immune complexes at the exterior of the tegumental membrane (Fig. 2b), which was rather similar to the aggregates of complexes seen in 28 day old parasites. In vitro excysted juvenile E. caproni were likewise free of immune complexes on the surface, and usually no material was observed outside the tegumental membrane, except for a thin regular glycocalyx (Fig. 2~). Incubation ofjuveniles in immune serum led to the formation of an irregular layer of immune complexes, as visualized by the ferritin labelling (Fig. 2d) showing
939
that even newly excysted juveniles were excreting antigens from the tegument. As in mature adult parasites, the tegument of juvenile and 7 day old E. cuproni was densely packed with membrane-bound vesicles (Fig. 2ad), which were of the same morphological types seen in the adult parasites. Despite examination of large numbers of parasite sections by transmission electron microscopy, we failed to find any vesicles fusing with the outer tegumental membrane in these younger stages of parasites. Cyst walls of E. caproni metacercariae were sometimes incidentally prepared together with the juvenile parasites. Only the inner lamellated layer of the cyst wall (Gulka & Fried, 1979) was present, and apparently the outer layers had been dissolved in the excystation process. Incubation of cyst walls in normal or immune mouse serum, followed by the ferritin labelling procedure, both led to intensive binding of ferritin on the most external lamellae. The binding of ferritin conjugate to the cyst walls was therefore nonspecific, and not due to the presence of antimetacercarial antibodies in immune mouse serum (data not shown). DISCUSSION Previous studies have shown that infections with E. caproni in mice induce a significant antibody response against the surface of the parasites (Simonsen & Andersen, 1986). The antibodies can be detected in serum as well as on the surface of parasites recovered from the intestinal lumen of mice from approximately 2 weeks after infection. However, the surface-bound antibodies are rapidly lost from the live parasite surface during culturing in vitro (Simonsen & Andersen, 1986). It has furthermore been shown that E. caproni surface antigens are continuously shed to the surroundings from both juvenile and adult parasites (Andresen et al., 1989). The results from the present study confirm the previous observations. The surface of adult E. cuproni recovered 28 days after infection of mice was covered by a thick irregular matrix containing mouse antibodies, as indicated by ferritin labelling. The mouse antibodies probably bound to the antigens which are secreted from the parasite surface (Andresen et al., 1989), thereby forming a layer of immune complexes. Culturing of the parasites in vitro led to loss of the immune complex covering. More antigens were continuously presented on the parasite surface,
FIG. 2. Transmission electron micrographs of the surface ofjuvenile and 7 day old E. cuproniafter treatment with murine sera and ferritin-conjugated antibody. F, ferritin molecules; IC, matrix of immune complexes; M, outer tegumental membrane; V, membrane-bound vesicle; S, spine. Scale bars = 400 nm. (a) Seven day old parasite freshly recovered from mouse. Note presence of glycocalyx and absence of immune complexes. (b) Seven day old parasite after incubation in immune mouse serum. Note presence of immune complexes. (c) In vitro excysted juvenile parasite. Note absence of immune complexes after incubation with normal mouse serum. (d) In vitro excysted parasite after incubation in immune mouse serum. Note presence of immune complexes indicating antigen on the surface of the parasite.
940
P. E. SIMONSEN,B. J. VENNERVALDand A. BIRCH-ANDERSEN
however, since re-incubation of the parasites in immune serum caused re-formation of the immune complex covering. Juvenile and young adult parasites, which had not previously been exposed to anti-surface antibodies, became covered with immune complexes after exposure to immune mouse serum. Juvenile and young adult parasites therefore also express antigens on their surface, which agrees with previous observations (Simonsen & Andersen, 1986; Andresen et al., 1989). Electron microscopic examination of juvenile Fasciola hepatica has also shown the formation of a matrix on the surface of the parasites when they were incubated in vitro in serum from immune sheep (Hanna, 1980a) or rats (Burden, Hughes & Hammet, 1982), and in vivo during infection in immune rats and mice (Burden, Bland, Hughes & Hammet, 1983). It was suggested in these studies that the matrix consisted of immune complexes. Hanna (1980a) furthermore demonstrated that antigens were continuously formed and sloughed from the surface ofjuvenile F. hepatica in vitro.
Apparently the E. caproni parasites were not harmed by the host’s antibody attack on the surface. The 28 day old adult parasites were active and producing eggs at the time of recovery from the mice (Odaibo et al., 1988), and no damage could be observed. The antigens seemed to be constituents of an irregular glycocalyx, or they were present as isolated excretions along the parasite surface. The anti-surface antibodies did not react with the parasite surface membrane, but formed complexes with antigens which were rather loosely attached to the outer surface of the parasites. The loose association of antigens to the parasite may have been the cause of the clumpy appearance of the matrix of immune complexes on the electron micrographs, since much of the material may have been lost in the long preparation process for electron microscopy. The use of a double sandwich technique for the demonstration of the surface antigens made the antigen-antibody complexes bigger but also more vulnerable to the preparation procedures. In our micrographs of thin sections of the organisms, tegumental vesicles from mature adult parasites were sometimes seen fused with the outer thus establishing direct tegumental membrane, contact to the exterior. Exocytosis of tegumental secretory products by fusing of vesicles with the outer tegumental membrane has been observed in other trematodes (Hanna, 1980b; Gress & Lumsden, 1976; Sharma & Hanna, 1988; Shannon & Bogitsh, 1971), as well as in cestodes (Oaks & lumsden, 1971; Lumsden, 1975). When the rapid turn-over of antigens on the E. caproni surface is considered (Simonsen & Andersen, 1986; Andresen et al., 1989), rather few vesicles were seen releasing their content to the exterior, and the actual event of exocytosis is possibly of extremely short duration. Matricon-Gondran (1980) and Andresen et al. (1989) identified two morphological types of membrane-bound vesicles in the tegument of E.
caproni, namely elongate (most common) and circulate (more rare). Careful examination of a large number of electron micrographs of juvenile and adult parasites in the present study confirmed the dominant presence of these two types in the two-dimensional pictures. However, for steric reasons, both types can be derived very well from a biconcave disc type of vesicle, depending on the angle at which the vesicle is sectioned, and it may be that only one type of vesicle is actually present in the distal cytoplasm of E. caproni. It is tempting to speculate on the role of the large amount of antigens excreted from the E. caproni surface. The material may simply be waste products of parasite metabolism, but it could also have an immuno-protective effect, e.g. by mechanically preventing antibodies from reaching the tegumental membrane, or by inducing immuno-suppressive reactions in the host. Recent studies have furthermore suggested that E. caproni parasites secrete vasoactive polypeptide-like components from the tegument (Thorndyke & Whitfield, 1987) which may affect the host’s intestinal physiology, and secreted antigens may also be responsible for the intensive pathological changes observed in the intestinal tissue of the infected host (Simonsen, Bindseil & Kie, 1989).
assistance of MS Helene Ravn, of Biophysics, Statens Serum Institut, in
Acknowledgements-The
Department
gratefully sectioning and electron microscopy is acknowledged, and MS Susan Wedel is similarly thanked for her photographic work. The studies were supported by grants from the Danish Natural Science Research Council. REFERENCES ANDRESEN K., SIMONSENP. E., ANDERSEN B. J. & BIRCHANDERSENA. 1989. Echinostoma caproni in mice: shedding of antigens from the surface of an intestinal trematode. International Journal for Parasiiology 19: 11 l-l 18. BURDEND. J.. HUGHES D. L. & HAMMETN. C. 1982. Fasciola hepatica: antibody coating of juvenile flukes in the intestinal lumen of resistant rats. Research in Veterinary Science 32: 4441. BURDEND. J., BLAND A. P., HUGHES D. L. & HAMMETN. C. 1983. Fasciola hepatica: development of the tegument of normal and y-irradiated flukes during infection in rats and mice. Parasitology 86: 137-145. CHRISTENSENN. O., KNUDSEN J. & ANDREASSENJ. 1986. Echinostoma revolutum: resistance to secondary and superimposed infections in mice. Experimental Parasitology 61: 311-31s. CHRISTENSENN. O., ODAIBO A. B. & SIMONSENP. E. 1988. Echinostoma population regulation in experimental rodent definitive hosts. Parasifology Research 15: 83-87. GRESS F. M. & LUMSDEN R. D. 1976. Ultrastructural cytochemistry of the tegument surface membrane of Paragonimus kellicotti. Rice University Studies 62: 11 l143. GULKA G. J. & FRIED B. 1979. Histochemical and ultrastructural studies on the metacercarial cyst of Echinostoma revolutum (Trematoda). International Journal for Parasitology 9: 57-59. HANNA R. E. B. 1980a. Fasciola hepatica: glycocalyx replacement in the juvenile as a possible mechanism for
Immune
complexes
protection against host immunity. Experimental Parasitology 50: 103- 114. HANNA R. E. B. 1980b. Fusciola hepatica: autoradiography of protein synthesis, transport and secretion by the tegument. Experimental Parasitology 50: 297-304. LUMSDEN R. D. 1975. Surface ultrastructure and cytochemistry of parasitic helminths. Experimental Parasitology 37: 267-339. MATRICON-GONDRAN M. 1980. Gap junctions and particle aggregates in the tegumentary syncytium of a trematode. Tissue & Cell 12: 383-394. OAKS J. & LUMSDEN R. 1971. Cytological studies on the absorptive surface of cestodes. V. Incorporation of carbohydrate-containing macromolecules into tegument membranes. Journal of Parasitology 51: 12561268. ODAIBO A. B., CHRISTENSENN. 0. & UKOLI F. M. A. 1988. Establishment, survival, and fecundity in Echinosfoma cuproni (Trematoda) infections in NMRI mice. Proceedings of the Helminthological Society of Washington 55: 265-269. ODAIBO A. B., CHRISTENSENN. 0. & UKOLI F. M. A. 1989. Further studies on the population regulation in Echinostoma caproni infections in NMRI mice. Proceedings of the
on E. cuproni surface
941
Helminthological
Society of Washington 56: 192-198. temperatus: radioautography of glucose-‘H and incorporation. Experimental Parasitology 29:
SHANNONW. & B~GIT~H B. 1971. Megalodiscus
comparative galactose-‘H 309-319. SHARMA P. N. & HANNA R. E. B. 1988. Ultrastructure and cytochemistry of the tegument of Orthocoelium scoliocoelium and Parumphistomum cervi (Trematoda: Digenea). Journal of Helminthology 62: 33 l-343. SIMONSEN P. E. & ANDERSEN B. J. 1986. Echinostoma revolutum in mice; dynamics of the antibody attack to the surface of an intestinal trematode. International Journalfor Parasitology 16: 475482. SIMONSENP. E., BINDSEILE. & K~IE M. 1989. Echinostoma caproni in mice: studies on the attachment site of an intestinal trematode. International Journalfor Parasitology 19: 561-566. THORNDYKE M. C. & WHITFIELD P. J. 1987. Vasoactive intestinal polypeptide-like immunoreactive tegumental cells in the digenean helminth Echinostoma fiei: possible role in host-parasite interactions. Generaland Comparative Endocrinology 68: 202-207.
Parasiloiogy
Vol. 20, No. 7. pp. 93S941.
1990 0
ECHINOSTOMA
CAPRONI
co2tL7519/90 $3.00 + 0.00 Pergamon Press p/c Socieryfor Pnrosiro/ogy
IN MICE: ULTRASTRUCTURAL
ON THE FORMATION OF IMMUNE COMPLEXES OF AN INTESTINAL TREMATODE PAUL E. SIMONSEN,*~
1990 Awmlim
B. JYDING
VENNERVALD*
STUDIES ON THE SURFACE
and A. BIRCH-ANDERSEN~
* Danish Bilharziasis Laboratory, Jaegersborg Alle 1 D, 2920 Charlottenlund, Denmark fDepartment of Biophysics, Statens Serum Institut, Amager Boulevard 80,230O Copenhagen S, Denmark (Received 23 March 1990; accepted 11 June 1990) AbStrzW-SIMONSEN P. E.,~ENNERVALD B.J. and BIRCH-ANDERSEN A. 1990.Echinostoma caproni in mice: ultrastructural studies on the formation of immune complexes on the surface of an intestinal trematode. International Journalfor Parasitology 20: 935-941. The binding of mouse antibodies to the surface antigens of juvenile and 7 and 28 day old Echinostoma caproni was examined by transmission electron microscopy of thin sections of parasites, which were treated with antibodies in a double sandwich technique with ferritinconjugated antibody. The surface of freshly recovered mature adult parasites was covered with an irregular but often rather intensive mouse antibody containing matrix, which probably represents a layer of mouse antibody/parasite antigen complexes. The complexes were lost after in vitro culturing of the parasites for 24 h, but incubation of the in vitro-maintained antibody-negative adult parasites with immune mouse serum led to reformation of a similar but less intensive cover with immune complexes. Juvenile and young stages of E. caproni, which had never been exposed to host antibodies, obtained a layer of immune complexes on their surface after incubation with immune mouse serum in vitro. In both young and mature parasites, the antibody-antigen complexes were observed to be rather loosely attached to the outer surface of the parasites, where the antigens probably constitute a part of the irregular glycocalyx of the organisms. It may also be that the antigens are present as isolated excretions along the surface of the parasites. Several sections indicated that the parasite surface antigens may be present in the tegument in vesicles which fuse with the outer membrane of the parasite whereby their contents are released to the exterior. INDEX KEY WORDS: Echinostoma caproni; intestinal trematode; mice; antibodies; immune complexes; ultrastructure; ferritin-conjugated antibody labelling; surface antigens; antigen shedding; tegumental
Infections with E. cuproni in mice induce a significant antibody response to the surface of the parasites (Simonsen & Andersen, 1986). The antibodies can be measured both in serum and on the surface of the parasites in the mouse intestine. Antibodies to the surface of juvenile parasites appear in serum at the same time as resistance to reinfection develops. However, antibodies which are bound to the live parasite surface are rapidly lost in vitro, in a process involving shedding of antigens from the parasites at a high rate (Simonsen & Andersen, 1986; Andresen, Simonsen, Andersen & Birch-Andersen, 1989). The effect of the antibodies on the E. caproni surface, and their significance in regulating the parasite population in the host, are presently not known. In the present investigation, the interaction between antigens on the surface of E. caproni and mouse antibodies was examined at the ultrastructural level, using a ferritin-conjugated antibody technique. The purpose was to localize surface antigens, and to visualize the dynamics of immune complex formation on the surface of E. caproni.
INTRODUCTION
MICE infected with Echinostoma caproni constitute a useful laboratory model for studies on intestinal trematodes in the final host (Christensen, Odaibo & Simonsen, 1988). Detailed observations have been documented on the parasite population regulation in this model (Christensen, Knudsen & Andreassen, 1986; Odaibo, Christensen & Ukoli, 1988, 1989). The cause of a primary E. caproni infection in mice is infection dose dependent. Light infections with six parasites are expelled in 68 weeks, whereas heavy infections with 25 parasites are not expelled. However, approximately 2 weeks after a primary infection of any size, complete resistance to reinfection develops, which seems to be of an immunological nature (Christensen et al., 1986). Resistance to reinfection does not affect the initial infection and therefore this host-parasite relationship represents an example of concomitant immunity.
t To whom all correspondence
should be addressed. 935
936
P. E. SIMONSEN, B. J. VENNERVALD and A. BIRCH-ANDERSEN MATERIALS
AND METHODS
Mice and parasites. Female
NMRI mice were obtained from Bomholtgaard Ltd, Denmark. Only mice more than 6 weeks old at the start of the experiments were used in these studies. Echinostoma caproni is identical to the population also named E. revolutum in previous work from the Danish Bilharziasis Laboratory (see Christensen et al., 1988). The life cycle is maintained in mice and Biomphalariu glubrata snails. Mice are infected by stomach tube feeding with metacercariae obtained by dissection of infected snails. Sera. Mouse blood samples were obtained by cardiac puncture under deep ether anaesthesia. Normal non-immune serum was obtained from uninfected mice, whereas immune serum was from a group of mice infected with 25 metacercariae 4 weeks previously. Preparation of juvenile parasites. Juvenile parasites were produced by in vitro excystation of metacercariae with trypsin and sodium taurocholate as previously described (Simonsen & Andersen, 1986). After excystation they were washed in PBS, pH 7.2, and prefixed in 2% formalin in PBS. Before serum incubations they were washed three times in PBS, once in 50 mM-NH,Cl and three times in PBS to block any reactive aldehyde groups. They were then divided into two groups which were incubated for 30 min with normal or immune mouse serum (1:20 in PBS), washed twice with PBS, incubated for 30 min with rabbit-anti-mouse antibody (1:40; Dakopatt), washed twice with PBS, incubated for 30 min with ferritin-conjugated goat-anti-rabbit IgG (1:25; MilesYeda Ltd, Israel) and finallv washed three times with PBS. Another batch ofjuvenile parasites was washed with PBS for removal of the excystation medium and then divided into two groups for incubation with immune or control serum in the living state, before they were killed with 3% glutaraldehyde in cacodylate buffer (pH 7.2) at room temperature for 30 min. No difference was noted between the preservation of ultrastructure or antigenicity of the organisms from the first and second batch, and consequently the results are not kept separate in the following experiments. Preparation of 7 day old parasites. The parasites were recovered from mice 7 days after infection with 25 metacercariae. After washing in PBS, they were incubated in normal or immune serum for 30 min, followed by washing three times in PBS. Further incubation in rabbit-anti-mouse antibody and ferritin-conjugated goat-anti-rabbit IgG was carried out as described for juvenile parasites. Finally, the parasites were killed by incubation with 3% glutaraldehyde in cacodylate buffer (pH 7.2) at room temperature for 30 min, before washing three times with PBS. Preparation of mature adult parasites. Mature adult parasites were obtained by dissection of mice 28 days after infection with 25 metacercariae. The parasites were washed rapidly in PBS. Adults for direct examination were then prefixed immediately with 2% formalin in PBS, washed three times in PBS, once in 50 mM-NH&l and three times in PBS. The parasites were then treated with rabbit-anti-mouse
antibody and ferritin-conjugated goat-anti-rabbit IgG, and washed in PBS as described for juvenile parasites. Other adults were maintained in vitro in RPM1 1640 tissue culture medium supplemented with 25 mM-HEPES, 22 mM-NaHCO, and 50 pg-mll ’ gentamycin (Garamycin, Schering Corp., U.S.A.) for 24 h at 37°C before prefixation with 2% fonnalin in PBS. After washing in PBS and 50 mM-NH,CI as above, these adults were incubated in normal or immune mouse serum (1:10 in PBS), rabbit-anti-mouse antibody and ferritin-conjugated goat-anti-rabbit IgG, before washing in PBS as described for iuvenile parasites. After the above preparations, all adult parasites were cut transversely while still soaked in PBS, and only the anterior third of the worms were further prepared for electron microscopy. Fixation and embedding for transmission electron microscopy. After the treatments described above, whole juvenile and 7 day old parasites, and the anterior third of adult parasites were transferred from the PBS to 45°C melted 1.5% Noble agar (Difco) in cacodvlate buffer pH 7.2. After cooling, small-blocks of worms embedded in the agar were fixed in 3% nlutaraldehvde in 0.1 M-cacodvlate buffer DH 7.2 at 4°C overnight. Furiher preparation for sectioning and electron microscopy followed the procedure previously described (Andresen ef al., 1989). RESULTS
adult parasites Freshly recovered 28 day old E. caproni, fixed immediately after recovery and incubated with ferritin-labelled conjugate, were examined in the transmission electron microscope. The parasites were covered with an irregular but often rather intensive matrix of material on the outer tegumental membrane (Fig. la). The presence and position of ferritin molecules in the matrix, together with the fact that it has previously been demonstrated that antigens are continuously excreted from the surface of live E. caproni (see Andresen et al., 1989), suggest that the matrix was made of complexes of parasite antigen and mouse antibody. The membrane itself was never labelled with ferritin, however, implying that parasite surface antigens are not integrated into the membrane, but rather seem to be intermingled with an irregular glycocalyx or excreted to the exterior from the surface of the parasites. Maintenance of the mature adult parasites for 24 h in vitro resulted in loss of the covering matrix from the surface of the parasites, and ferritin labelling was no longer present (Fig. lb). Only a thin regular layer of glycocalyx was observed on the outside of the tegumental membrane. The immune complexes which were present qn freshly recovered parasites had Mature
FIG. 1. Transmission electron micrographs of the surface of 28 day old E. caproni, after treatment with murine sera and ferritinconjugated antibody. F, ferritin molecules; IC, matrix of immune complexes; M, outer tegumental membrane; V, membranebound vesicle. Scale bars = 400 nm. (a) Freshly recovered parasite. The arrow indicates a vesicle which is fused with the outer tegumental membrane. (b) Parasite after 24 h of in vitro maintenance followed by incubation in normal mouse serum. Note absence of immune complexes. (c)Parasite after 24 h of in vitro culture followed by incubation in immune mouse serum. Note presence of immune complexes. The arrow indicates a vesicle which is fused with the outer tegumental membrane, thus establishing direct contact with the exterior.
Immune complexes on E. cuproni surface
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Immune complexes on E. cuproni surface apparently been sloughed off during the in vitro culture. In vitro exposure of the cultured antibody-negative adult parasites to immune mouse serum led to reformation of immune complexes on the outside of the parasites (Fig. lc). Although large amounts of antigen/antibody complexes were present on the parasite surface, the aggregates were usually smaller and the covering less intensive than seen with freshly recovered adult parasites. The re-formation of immune complexes after shedding of the original complexes indicates that antigenic material must be continuously released from the tegument to the parasite surface and further to the exterior. The tegument of all 28 day old E. cuproni was densely packed with membrane-bound vesicles, especially near the outer tegumental membrane (Fig. la-c), indicating an active secreting surface. The morphology of the vesicles was rather variable, but most of them belonged to the circular or elongate types described previously (Andresen et al., 1989). Occasionally vesicles were seen to fuse with the outer tegumental membrane (Fig. la and c), and thereby releasing their contents to the exterior. At least part of the released material was antigenic, as indicated by the ferritin labelling. Younger stages Seven day old E. cuproni, freshly recovered from mouse intestines, exhibited no immune complexes on their surface, when examined by the ferritin labelling technique (Fig. 2a). This result is not surprising, since antibodies against the parasite surface are not produced by mice 7 days after a primary infection (Simonsen & Andersen, 1986). Some cloudy loose material was observed exterior to the outer leaflet of the tegumental membrane, which may have been uncomplexed antigens. Incubation of the parasites in immune mouse serum led to the formation of an irregular but intensive layer of immune complexes at the exterior of the tegumental membrane (Fig. 2b), which was rather similar to the aggregates of complexes seen in 28 day old parasites. In vitro excysted juvenile E. caproni were likewise free of immune complexes on the surface, and usually no material was observed outside the tegumental membrane, except for a thin regular glycocalyx (Fig. 2~). Incubation ofjuveniles in immune serum led to the formation of an irregular layer of immune complexes, as visualized by the ferritin labelling (Fig. 2d) showing
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that even newly excysted juveniles were excreting antigens from the tegument. As in mature adult parasites, the tegument of juvenile and 7 day old E. cuproni was densely packed with membrane-bound vesicles (Fig. 2ad), which were of the same morphological types seen in the adult parasites. Despite examination of large numbers of parasite sections by transmission electron microscopy, we failed to find any vesicles fusing with the outer tegumental membrane in these younger stages of parasites. Cyst walls of E. caproni metacercariae were sometimes incidentally prepared together with the juvenile parasites. Only the inner lamellated layer of the cyst wall (Gulka & Fried, 1979) was present, and apparently the outer layers had been dissolved in the excystation process. Incubation of cyst walls in normal or immune mouse serum, followed by the ferritin labelling procedure, both led to intensive binding of ferritin on the most external lamellae. The binding of ferritin conjugate to the cyst walls was therefore nonspecific, and not due to the presence of antimetacercarial antibodies in immune mouse serum (data not shown). DISCUSSION Previous studies have shown that infections with E. caproni in mice induce a significant antibody response against the surface of the parasites (Simonsen & Andersen, 1986). The antibodies can be detected in serum as well as on the surface of parasites recovered from the intestinal lumen of mice from approximately 2 weeks after infection. However, the surface-bound antibodies are rapidly lost from the live parasite surface during culturing in vitro (Simonsen & Andersen, 1986). It has furthermore been shown that E. caproni surface antigens are continuously shed to the surroundings from both juvenile and adult parasites (Andresen et al., 1989). The results from the present study confirm the previous observations. The surface of adult E. cuproni recovered 28 days after infection of mice was covered by a thick irregular matrix containing mouse antibodies, as indicated by ferritin labelling. The mouse antibodies probably bound to the antigens which are secreted from the parasite surface (Andresen et al., 1989), thereby forming a layer of immune complexes. Culturing of the parasites in vitro led to loss of the immune complex covering. More antigens were continuously presented on the parasite surface,
FIG. 2. Transmission electron micrographs of the surface ofjuvenile and 7 day old E. cuproniafter treatment with murine sera and ferritin-conjugated antibody. F, ferritin molecules; IC, matrix of immune complexes; M, outer tegumental membrane; V, membrane-bound vesicle; S, spine. Scale bars = 400 nm. (a) Seven day old parasite freshly recovered from mouse. Note presence of glycocalyx and absence of immune complexes. (b) Seven day old parasite after incubation in immune mouse serum. Note presence of immune complexes. (c) In vitro excysted juvenile parasite. Note absence of immune complexes after incubation with normal mouse serum. (d) In vitro excysted parasite after incubation in immune mouse serum. Note presence of immune complexes indicating antigen on the surface of the parasite.
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however, since re-incubation of the parasites in immune serum caused re-formation of the immune complex covering. Juvenile and young adult parasites, which had not previously been exposed to anti-surface antibodies, became covered with immune complexes after exposure to immune mouse serum. Juvenile and young adult parasites therefore also express antigens on their surface, which agrees with previous observations (Simonsen & Andersen, 1986; Andresen et al., 1989). Electron microscopic examination of juvenile Fasciola hepatica has also shown the formation of a matrix on the surface of the parasites when they were incubated in vitro in serum from immune sheep (Hanna, 1980a) or rats (Burden, Hughes & Hammet, 1982), and in vivo during infection in immune rats and mice (Burden, Bland, Hughes & Hammet, 1983). It was suggested in these studies that the matrix consisted of immune complexes. Hanna (1980a) furthermore demonstrated that antigens were continuously formed and sloughed from the surface ofjuvenile F. hepatica in vitro.
Apparently the E. caproni parasites were not harmed by the host’s antibody attack on the surface. The 28 day old adult parasites were active and producing eggs at the time of recovery from the mice (Odaibo et al., 1988), and no damage could be observed. The antigens seemed to be constituents of an irregular glycocalyx, or they were present as isolated excretions along the parasite surface. The anti-surface antibodies did not react with the parasite surface membrane, but formed complexes with antigens which were rather loosely attached to the outer surface of the parasites. The loose association of antigens to the parasite may have been the cause of the clumpy appearance of the matrix of immune complexes on the electron micrographs, since much of the material may have been lost in the long preparation process for electron microscopy. The use of a double sandwich technique for the demonstration of the surface antigens made the antigen-antibody complexes bigger but also more vulnerable to the preparation procedures. In our micrographs of thin sections of the organisms, tegumental vesicles from mature adult parasites were sometimes seen fused with the outer thus establishing direct tegumental membrane, contact to the exterior. Exocytosis of tegumental secretory products by fusing of vesicles with the outer tegumental membrane has been observed in other trematodes (Hanna, 1980b; Gress & Lumsden, 1976; Sharma & Hanna, 1988; Shannon & Bogitsh, 1971), as well as in cestodes (Oaks & lumsden, 1971; Lumsden, 1975). When the rapid turn-over of antigens on the E. caproni surface is considered (Simonsen & Andersen, 1986; Andresen et al., 1989), rather few vesicles were seen releasing their content to the exterior, and the actual event of exocytosis is possibly of extremely short duration. Matricon-Gondran (1980) and Andresen et al. (1989) identified two morphological types of membrane-bound vesicles in the tegument of E.
caproni, namely elongate (most common) and circulate (more rare). Careful examination of a large number of electron micrographs of juvenile and adult parasites in the present study confirmed the dominant presence of these two types in the two-dimensional pictures. However, for steric reasons, both types can be derived very well from a biconcave disc type of vesicle, depending on the angle at which the vesicle is sectioned, and it may be that only one type of vesicle is actually present in the distal cytoplasm of E. caproni. It is tempting to speculate on the role of the large amount of antigens excreted from the E. caproni surface. The material may simply be waste products of parasite metabolism, but it could also have an immuno-protective effect, e.g. by mechanically preventing antibodies from reaching the tegumental membrane, or by inducing immuno-suppressive reactions in the host. Recent studies have furthermore suggested that E. caproni parasites secrete vasoactive polypeptide-like components from the tegument (Thorndyke & Whitfield, 1987) which may affect the host’s intestinal physiology, and secreted antigens may also be responsible for the intensive pathological changes observed in the intestinal tissue of the infected host (Simonsen, Bindseil & Kie, 1989).
assistance of MS Helene Ravn, of Biophysics, Statens Serum Institut, in
Acknowledgements-The
Department
gratefully sectioning and electron microscopy is acknowledged, and MS Susan Wedel is similarly thanked for her photographic work. The studies were supported by grants from the Danish Natural Science Research Council. REFERENCES ANDRESEN K., SIMONSENP. E., ANDERSEN B. J. & BIRCHANDERSENA. 1989. Echinostoma caproni in mice: shedding of antigens from the surface of an intestinal trematode. International Journal for Parasiiology 19: 11 l-l 18. BURDEND. J.. HUGHES D. L. & HAMMETN. C. 1982. Fasciola hepatica: antibody coating of juvenile flukes in the intestinal lumen of resistant rats. Research in Veterinary Science 32: 4441. BURDEND. J., BLAND A. P., HUGHES D. L. & HAMMETN. C. 1983. Fasciola hepatica: development of the tegument of normal and y-irradiated flukes during infection in rats and mice. Parasitology 86: 137-145. CHRISTENSENN. O., KNUDSEN J. & ANDREASSENJ. 1986. Echinostoma revolutum: resistance to secondary and superimposed infections in mice. Experimental Parasitology 61: 311-31s. CHRISTENSENN. O., ODAIBO A. B. & SIMONSENP. E. 1988. Echinostoma population regulation in experimental rodent definitive hosts. Parasifology Research 15: 83-87. GRESS F. M. & LUMSDEN R. D. 1976. Ultrastructural cytochemistry of the tegument surface membrane of Paragonimus kellicotti. Rice University Studies 62: 11 l143. GULKA G. J. & FRIED B. 1979. Histochemical and ultrastructural studies on the metacercarial cyst of Echinostoma revolutum (Trematoda). International Journal for Parasitology 9: 57-59. HANNA R. E. B. 1980a. Fasciola hepatica: glycocalyx replacement in the juvenile as a possible mechanism for
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protection against host immunity. Experimental Parasitology 50: 103- 114. HANNA R. E. B. 1980b. Fusciola hepatica: autoradiography of protein synthesis, transport and secretion by the tegument. Experimental Parasitology 50: 297-304. LUMSDEN R. D. 1975. Surface ultrastructure and cytochemistry of parasitic helminths. Experimental Parasitology 37: 267-339. MATRICON-GONDRAN M. 1980. Gap junctions and particle aggregates in the tegumentary syncytium of a trematode. Tissue & Cell 12: 383-394. OAKS J. & LUMSDEN R. 1971. Cytological studies on the absorptive surface of cestodes. V. Incorporation of carbohydrate-containing macromolecules into tegument membranes. Journal of Parasitology 51: 12561268. ODAIBO A. B., CHRISTENSENN. 0. & UKOLI F. M. A. 1988. Establishment, survival, and fecundity in Echinosfoma cuproni (Trematoda) infections in NMRI mice. Proceedings of the Helminthological Society of Washington 55: 265-269. ODAIBO A. B., CHRISTENSENN. 0. & UKOLI F. M. A. 1989. Further studies on the population regulation in Echinostoma caproni infections in NMRI mice. Proceedings of the
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comparative galactose-‘H 309-319. SHARMA P. N. & HANNA R. E. B. 1988. Ultrastructure and cytochemistry of the tegument of Orthocoelium scoliocoelium and Parumphistomum cervi (Trematoda: Digenea). Journal of Helminthology 62: 33 l-343. SIMONSEN P. E. & ANDERSEN B. J. 1986. Echinostoma revolutum in mice; dynamics of the antibody attack to the surface of an intestinal trematode. International Journalfor Parasitology 16: 475482. SIMONSENP. E., BINDSEILE. & K~IE M. 1989. Echinostoma caproni in mice: studies on the attachment site of an intestinal trematode. International Journalfor Parasitology 19: 561-566. THORNDYKE M. C. & WHITFIELD P. J. 1987. Vasoactive intestinal polypeptide-like immunoreactive tegumental cells in the digenean helminth Echinostoma fiei: possible role in host-parasite interactions. Generaland Comparative Endocrinology 68: 202-207.