Clin. exp. Immunol. (1976) 26, 181-187.
Acquisition of human blood group antigens by Schistosoma mansoni 0. L. GOLDRING, *J. A. CLEGG, S. R. SMITHERS & R. J. TERRY National Institute for Medical Research, Mill Hill, London and Brunel University, Uxbridge, Middlesex (Received 1 March 1976) SUMMARY
Juvenile forms of Schistosoma mansoni (schistosomula) have been cultured in human blood of various specificities and tested for the presence of blood group substances on their surfaces. The tests employed were survival following transfer into rhesus monkeys immunized against human blood substances, mixed agglutination reactions, and immunofluorescence. A, B, H and Lewisb+ antigens were expressed at the surface when the parasites were cultured in blood of appropriate specificities. Rhesus, M N S, and Duffy antigens could not be detected on the parasite surface following culture. The evidence suggests that the expressed blood group antigens are of host origin and are acquired by the parasite during culture, probably in the form of glycolipids or megaloglycolipids. It is likely that these substances are also acquired by parasites in the bloodstream of man. They may serve to mask surface parasite antigens, and so enable schistosomes to evade parasitespecific humoral or cellular immune responses. INTRODUCTION A close and intimate association between host-like antigenic determinants and the surface of the trematode parasite S. mansoni was first demonstrated by Smithers, Terry & Hockley (1969). Schistosomes grown to maturity in mice survived well on transfer into the portal systems of rhesus monkeys, but were rapidly destroyed if these monkeys had first been immunized against mouse erythrocytes. The schistosomes were killed by antibodies directed against sites on the parasite surface which presumably shared antigen specificity with mouse erythrocytes. The existence of this close association of host-like antigenic determinants and schistosome surfaces has now been amply confirmed (Clegg, Smithers & Terry, 1970; 1971a and b; Cioli and Neis (1972); Perez & Terry, 1973; McLaren, Clegg & Smithers, 1975). It has been suggested that these determinants are truly of host origin, and that their acquisition and disposition at the parasite surface enables the adult parasite to evade the efferent arm of the host immune response (Clegg, 1974; Terry & Smithers, 1975). The evolution of such a protective device might explain the long survival of adult schistosomes in the blood of experimental animals and man. An understanding of the nature and mode of acquisition of host antigens by schistosomes might well be essential for any rational progress towards the development of vaccines against thesef important parasites. Clegg, Smithers & Terry (1971b) grew juvenile schistosomes (schistosomula) in culture media containing human blood of various specificities, and transferred them into the portal systems of monkeys immunized against human erythrocytes. These preliminary experiments suggested that the schistosomes acquired antigens during culture, some of which showed A or B blood group specificity. The aims of Present address: Wellcome Research Laboratories, Beckenham, Kent. Correspondence: Dr S. R. Smithers, National Institute for Medical Research, Mill Hill, London N.W.7. *
181
0. L. Goldring et al.
182
the present investigation were to identify exactly which human blood group antigens were acquired during culture and to determine in what form they were acquired.
MATERIALS AND METHODS Parasite. A Puerto Rican strain of S. mansoni was used throughout this work; its maintenance has been described previously (Smithers & Terry, 1965). Human blood. Four human bloods were used in this investigation; blood group antigen specificities are shown in Table 1. TABLE 1. Blood group specificities of human bloods used for the cultivation of
schistosomula ABO
C
A1 B 0 A1B
-
+
+
+
-
+
-
+ +
+ +
-
+
+ +
+ +
+ -
-
-
-
+
+
+
-
D
E
c
e
M
N
Fya
Leb
+
+
+
+ +
+ -
+ +
+
-
+
S
Human blood group substances. Purified human A blood group glycoprotein was a gift from Professor Winifred Watkins (Lister Institute of Preventive Medicine). Crude A, blood group 'megalolipid' was prepared from A1 cells by the methods of Gardas & Koscielak (1973 and 1974) and lyophilised. Monospecific blood group antisera. All sera were inactivated at 560C for 30 min. The following were obtained commercially from Ortho Pharmaceuticals: human anti A; B; rhesus C, c, D, E, and e; Duffy Fya+; S; rabbit anti-M; N; and Lewisb+. Other sera employed were a rabbit anti-blood group H substance, absorbed with human AIB erythrocytes (gift of Professor Watkins); and a rabbit anti-human globulin (Gift of Dr K. L. G. Goldsmith, MRC Blood Group Reference Laboratory). Normal rabbit serum absorbed with A1B erythrocytes provided the control serum. Fluorescein-labelled antisera. The following were obtained from Miles Laboratories: IgG fraction of FITC-labelled rabbit anti-human IgG; IgG fraction of FITC-labelled rabbit anti-human IgM; IgG fraction of FITC-labelled goat anti-rabbit IgG. Immunization ofmonkeys. Rhesus monkeys weighing between 2-6 and 4.5 Kg were each immunized with 0 3 mg of purified A glycoprotein emulsified in Freund's complete adjuvant (FCA). Four weeks later booster inoculations were given using the same immunization dose but without adjuvant. Inoculations were given both s.c. and i.m. in several sites. Cultivation ofschistosomula in human blood. Schistosomula were collected by allowing cercariae to penetrate isolated mouse skin and cultured in vitro for periods of up to 15 days, according to the methods of Clegg & Smithers (1972). The culture medium consisted of equal volumes of fresh human or monkey serum, and Earle's balanced salt solution with 0 5% lactalbumin hydrolysate and antibiotics (ELac). To this were added human or monkey erythrocytes, at a final concentration of 1%, or blood group substances at the concentrations specified in the descriptions of the experiments. The medium was gassed with 8% CO2 in air and the pH maintained at 7-4. The schistosomula were cultured in Leighton tubes, each containing 200-250 schistosomula at 37°C. The medium was changed every 3-4 days but the original erythrocyte inocula were retained. Satisfactory growth was judged by the criteria of Clegg (1965) which were established from in vivo observations. Transfer ofschistosomula to monkeys. Schistosomula for transfer were washed in ELac containing 1%/ normal monkey serum and then transferred into the portal systems of normal and immunized monkeys by cannulation of the mesenteric veins (Smithers & Terry, 1967). Five weeks later, when surviving worms had reached maturity, the monkeys were killed, perfused and the adult worms recovered (Smithers & Terry, 1965). Mixed agglutination. The principle of this test (Coombs, Bedford & Rouillard, 1956) is the formation of mixed aggregates of two cell types by antibody reacting with similar antigenic determinants occurring on the surfaces of the different cells. Fifteen-day schistosomula were removed from culture and washed several times with ELac containing 1% BSA (ELacBSA). They were allowed to settle, the washing fluid was removed and replaced by 50 ul of the appropriate antiserum. The schistosomula were incubated at 20°C for 1 hr, washed three times with ELac-BSA and then placed in 041 ml of a 2% erythrocyte suspension in ELac-BSA. The preparation, containing 10-30 schistosomula, was transferred to a microscope slide and examined for mixed agglutination with a Leitz inverted microscope. Positive and negative reactions are shown in Plates la and lb respectively. Immunofluorescence. The indirect method was employed. Schistosomula were well washed with ELac-BSA, and after
Human blood group antigens and S. mansoni
183
concentration 50 pl of the appropriate antiserum was added. They were incubated at room temperature for 30 min and then washed three times with ELac-BSA. 50 p1 of the appropriate undiluted FITC-antibody was added and the schistosomula incubated for a further 30 min at 20'C. The schistosomula were washed four times in ELac-BSA, transferred to a microscope slide and examined for fluorescence with a Reichert Binolux microscope; Wotan HB 200 W mercury vapour lamp; BG 12/5-2 mm exciter and Ilford 107 barrier filters.
RESULTS (a) In vivo experiments: expression of A substance Six rhesus monkeys were each immunized with purified A glycoprotein in FCA and four monkeys were inoculated with FCA alone. Booster inoculations were given 30 days later. Schistosomula were cultured for 15 days in either A 'secretor' blood, A 'non-secretor' blood or B 'secretor' blood. One week after the booster inoculation, these schistosomula were surgically transferred into the mesenteric veins of immunized and control monkeys as shown in Table 2. The numbers transferred varied between 100 and 150, but were accurately known for each transfer. Care was taken TABLE 2. Survival of S. mansoni in monkeys immunized against blood group substance A
a
Monkey
Immunization
Blood culture
Percentage of worm recovery
700 701 706 707 702 705 708 709 703 704
Blood group Substance A+FCA FCA FCA Blood group Substance A+FCA FCA FCA Blood group Substance A+FCA
A secretor A secretor A secretor A secretor A non-secretor A non-secretor A non-secretor A non-secretor B secretor B secretor
1 3 81 79 5 0 48 69 80 79
b
__ _ ~~~~~~~~~~~.."...................... t ~ .+ !. Am-| As
FIG. 1. (a) Positive mixed agglutination between 15-day schistosomulum (anterior end) and erythrocytes (Magnification x 720); (b), negative mixed agglutination (Magnification x 600).
"
184
0. L. Goldring et al.
to ensure that only fully viable and well-developed schistosomula were transferred. Five weeks later, the monkeys were killed, and the adult schistosomes were recovered by perfusion and counted. The results summarized in Table 2 are very clear. Recoveries from control monkeys, inoculated with FCA alone are high, validating the transfer and recovery procedures. Schistosomula cultured in A blood, either 'secretor' or 'non-secretor' were almost totally destroyed on transfer into anti-A monkeys, whereas those cultured in B 'secretor' blood survived normally. It is inferred that the schistosomula grown in A group blood, irrespective of secretor status, had A antigens on their surfaces. Those schistosomula which were grown in B blood did not possess A antigen. It is therefore suggested that those schistosomula which possessed A antigens had acquired them from the blood in which they had been grown.
(b) In vitro experiments The foregoing experiment had suggested the acquisition of A antigens by schistosomula grown in A blood. Unfortunately experiments of this type could not be used to study the acquisition of B and H blood group antigens as rhesus monkeys are known to possess B and H type substances in their saliva, although not on their erythrocytes, and so could not be immunized against them (Wiener, MoorJankowski & Socha, 1972). The possible acquisition of these antigens had perforce to be studied by in vitro methods using mixed agglutination and fluorescent antibodies. (1) Expression of blood group substances A, B and H as determined by mixed agglutination reactions. Schistosomula were cultured in human bloods of specificities A, B, 0 and AB for 15 days. At the end of this period, the schistosomula were reacted with antisera to blood group substances A, B and H and the indicator erythrocytes, and examined for mixed agglutination. Table 3 summarizes the design of this experiment and the results obtained. Positive reactions were seen where the blood used in culture and the indicator erythrocytes shared the same blood group specificity, and where the antiserum was directed against that specificity. Thus, schistosomula expressed only A antigen when cultured in A blood; only B antigen when cultured in B blood; only H antigen when cultued in 0 blood; and both A and B antigens when cultured in AB blood. (2) Expression of blood group substances A and B when cultured in serum. This experiment was designed to determine whether schistosomula acquire the blood group antigens directly from erythrocytes, or whether they can acquire them from serum. Glycoproteins with A B H specificity are found in the serum and other body fluids of 'secretors' but not 'non-secretors'; glycolipids with this specificity may occur in the sera of both (Gardas & Koscielak, 1973). Accordingly, schistosomula were cultured for 15 days in B serum, A 'secretor' serum or A 'nonsecretor' serum. The various sera were cleared from possible contamination with erythrocyte ghosts by centrifugation at 11,000 g for 30 mins. As schistosomula require erythrocytes for normal development O cells were added to all cultures. The schistosomula were then tested for the acquisition of A and B antigens by mixed agglutination. The plan and results of this experiment are summarized in Table 4. Schistosomula acquired B antigen when grown in B serum, and A antigen when grown in A serum, irrespective of secretor status. Seemingly schistosomula can acquire blood group antigens of A B H type from serum, and these antigens are likely to be in the form of glycolipids. (3) Acquisition of A blood group substance from an A 'megaloglycolipid' preparation. Gardas & Koscielak (1973 and 1974) have described water-soluble antigens of the human erythrocyte membrane with A, B, H and I activity. Chemical evidence suggests that these antigens are complex glycolipids, containing 20-40 sugar residues per mole of ceramide; these glycolipids have been designated 'megaloglycolipids'. Since we had some indirect evidence that schistosomula might be acquiring glycolipids from serum, we investigated their capacity to take up megalolipids prepared from A erythrocytes. Schistosomula were cultured in either B blood, or B blood to which had been added A megaloglycolipid at a concentration of 90 pg/ml. They were then examined for mixed agglutination with the appropriate antisera and indicator cells. All schistosomula were positive when tested with anti-B serum and B erythrocytes, and additionally, those cultured in the presence of A megaloglycolipid were positive
185
Human blood group antigens and S. mansoni TABLE 3. Detection of A, B and H antigens on schistosomula by mixed agglutination
Blood in culture
Antiserum
Indicator erythrocytes
No. of schistosomula observed
A B 0 A B 0 A B 0 A B 0 A B 0 A B 0 A B 0 A B 0
10 8 9 11 8 7 9 7 10 9 8 6 9 8 7 8 11 11 11 10 10 10 14 8
anti-A anti-B anti-H
A
B
*NRS anti-A anti-B anti-H
0
NRS anti-A anti-B anti-H
AB
NRS anti-A anti-B anti-H NRS
Percentage showing mixed agglutination 100 0 0 0 0 0 0 100 0 0 0 0 0 0 100 0 0 0 100 100 0 0 0 0
The 0 indicator erythrocytes were Lea /Leb- in order to eliminate possible reactions of Lewis antigens with the rabbit anti-H serum. * Normal rabbit serum absorbed with A1B erythrocytes.
TABLE 4. Detection of A and B antigens on schistosomula cultured in A and B serum
Culture Serum
Cells
B Secretor
0
A Secretor
0
A
0
Non-secretor
Antiserum
Indicator erythrocytes
No. of schistosomula observed
Anti-A Anti-B -
A B A B
14 8 9 12
0 100 0 0
Anti-A Anti-B
A B A B A B
12 11 7 8 14 11
100 0 0 0 100 0
Anti-A Anti-B
Percentage showing mixed agglutination
186
0. L. Goldring et al.
when tested with anti-A serum and A cells. Evidently schistosomula can acquire blood group antigens which are in megaloglycolipid form. (4) Failure to detect expression of M and N antigens by mixed agglutination. M and N antigens may be detected by direct erythrocyte agglutination with rabbit anti-M and anti-N antisera, containing specific IgM antibodies. Accordingly, schistosomula were cultured for 15 days in B type blood where the cells were M and N positive. They were then tested for their capacity to produce mixed agglutination reactions with M and N positive cells in the presence of rabbit anti-M and anti-N antisera. No positive reactions were observed, indicating that schistosomula do not acquire M and N antigens in culture. (5) Investigations on the expression of blood group antigens using immunofluorescence. Schistosomula were grown for 15 days in a range of human bloods, various members of which were positive for the A, B, S, rhesus (C.D.E.c.e), Lewis (Leb)+ and Duffy (Fy')+ blood group antigens (Table 1). The schistosomula were reacted with the relevant blood grouping sera, then treated with FITC-labelled antisera to human and rabbit immunoglobulins as appropriate. The usual controls were included. Strong positive specific immunofluorescence was seen on testing for A and B antigens, and weak reactions for the Lewis antigen. Tests for all other antigens were negative. We did not use antigenpositive red cells as positive immunofluorescence controls because of reported difficulties associated with the quenching effect of haemoglobin (Nairn, 1969). The activity of all serological reagents was however demonstrated by antiglobulin (Coombs) tests on antigen-positive red cells. The immunofluorescence studies therefore provide additional evidence that A and B antigens can be acquired by schistosomula in culture; there is evidence that Lewis`+ antigen may be acquired, but no evidence that the S, rhesus and Duffy antigens are acquired. DISCUSSION These experiments provide clear evidence for the presence of A, B and H substances on the surfaces of schistosomes cultured in human blood. The presence of a particular specificity on the parasite surface depends on that specificity being present in the blood used for culture. The simplest view is that the A, B and H substances are of host origin and are acquired by the parasite, but other explanations are not necessarily excluded by the data. It is perhaps surprising that the schistosomes seemed to acquire H antigen only when they were grown in 0 blood, as H antigen is also present on A, B and AB erythrocytes. It is known, however, that the amount of H antigen is less on these cells than on 0 cells, and it is possible that the more abundant A and B antigens compete strongly for sites on the parasite surface. Regarding other blood group antigens, there is preliminary evidence for the acquisition of Lewis (Leb+) antigen, but no evidence for the acquisition of M, N,S, rhesus and Duffy antigens. Our experiments indicated that schistosomes could acquire A and B antigens from the sera of A and B positive donors, in the absence of A and B erythrocytes. This occurred irrespective of the secretor status of the donor. Non-secretors do not possess the soluble glycoprotein A B H antigens in their body fluids, so that it seems unlikely that the antigens acquired by the parasites from serum was in the form of glycoproteins. High-speed centrifugation of the sera used for culture removed erythrocytes and ghosts. Nevertheless, it is possible that some membrane fragments remained in the sera, and that these provided the source of antigens. An alternative view, is that A B H substances may occur in the plasma of both secretors and non-secretors in the form of the megaloglycolipids described by Gardas & Koscielak (1973 and 1974). These workers showed that the glycolipids had the unique property of combining with cells and conferring upon them appropriate surface antigenic specificity. Further, they believe that these megaloglycolipids, present in the erythrocyte membrane, might be originally derived from the blood plasma. Our experiments have shown that culture in A megaloglycolipid conferred A specificity on the schistosome surface. In the absence of contrary evidence, it seems not unreasonable to believe that the A B H blood group specific substances present at the surface of schistosomes cultured in human blood, are derived from the plasma, and that antigens are similarly acquired in the bloodstream of man. Some support for this view is provided by the studies of other blood group antigens. Lewis substance
Human blood group antigens and S. mansoni
187
is acquired by schistosomes, and this is known to exist as a glycolipid in plasma and to have the property of attaching to cells (Marcus & Cass, 1969). The antigens of other groups, M, N, S, rhesus and Duffy, are believed to be glycoproteins, confined to the erythrocyte membrane, and these are not acquired by the parasite. These investigations provide no direct support for the view that the acquisition of host antigens enables the schistosome to evade host immunity (Clegg, 1974; Terry & Smithers, 1975). But it is difficult to escape the conclusion that the acquisition of substantial amounts of host blood group substances will serve to mask target antigens on the parasite surface and thus reduce the effectiveness of specific humoral or cellular immunity. We are grateful to Miss Elaine Holder and Mr K. Broomfield for their skilled technical assistance.
REFERENCES CIOLI, D. & NEIS, R. (1972) Antigeni dell'ospite in Schistosoma mansoni. Parassitologia, 14, 73. CLEGG, J.A. (1965) In vitro cultivation of Schistosoma mansoni. Exp. Parasit. 16, 133. CLEGG, J.A. (1974) Host antigens and the immune response in schistosomiasis. Parasites in the immunized host: mechanisms of survival, CIBA Foundation Symposium, 25 (new series), 161. CLEGG, J.A. & SMiTnERs, S.R. (1972) The effects of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro. Int. J. Parasit. 2, 79. CLEGG, J.A., SMITHERS, S.R. & TERRY, R.J. (1970) 'Host' antigens associated with schistosomes: observations on their attachment and their nature. Parasitology, 61, 87. CLEGG, J.A., SMITHERS, S.R. & TERRY, R.J. (1971a) Concomitant immunity and host antigens associated with schistosomiasis. Int. J. Parasit. 1, 43. CLEGG, J.A., SMITHERS, S.R. & TERRY, R.J. (1971b) Acquisition of human antigens by Schistosoma mansoni during cultivation in vitro. Nature (Lond.), 232, 653. COOMBS, R.R.A., BEDFORD, D. & ROUILLARD, L.M. (1956) A and B blood group antigens on human epidermal cells demonstrated by mixed agglutination. Lancet, i, 461. GARDAS, A. & KoscIELAK, J. (1973) New form of A-, Band H-blood group active substances extracted from erythrocyte membranes. Europ. J. Biochem. 32, 178. GARDAS, A. & KOSCIELAK, J. (1974) Megaloglycolipidsunusually complex glycosphingolipids of human erythrocyte membranes with A, B, H and I blood group specificity. FEBS letters, 42, 101.
McLAREN, D.J., CLEGG, J.A. & SMITHERS, S.R. (1975) Acquisition of host antigens by young Schistosoma mansoni in mice: correlation with failure to bind antibody in vitro. Parasitology, 70, 67. MARCUS, D.M. & CASS, L.E. (1969) Glycosphingolipids with Lewis bloodgroup activity; uptake by human erythrocytes. Science, 164, 553. NAIRN, R.C. (1969) Fluorescent protein tracing, 3rd edn (Ed. by R.C. Nairn), E. & S. Livingstone Ltd. Edinburgh and London. PEREZ, H. & TERRY, R.J. (1973) The killing of adult Schistosoma mansoni in vitro in the presence of antisera to host antigenic determinants and peritoneal cells. Int. 5. Parasit. 3, 499. SMITHERS, S.R. & TERRY, R.J. (1965) The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of the adult worms. Parasitology, 55, 695. SMITHERS, S.R. & TERRY, R.J. (1967) Resistance to experimental infections with Schistosoma mansoni in rhesus monkeys induced by the transfer of adult worms. Trans. roy. Soc. trop. Med. Hyg. 61, 517. SMITHERS, S.R., TERRY, R.J. & HOCKLEY, D.J. (1969) Host antigens in schistosomiasis. Proc. roy Soc. B. 171, 483. TERRY, R.J. & SMITHERS, S.R. (1975) Evasion ofthe immune response by parasites. Symposia Soc. exp. Biol. 29, 453. WEINER, A.S., MOOR-JANKOWSKI, J. & SOCHA, W.W. (1972) Principles of blood grouping in apes and monkeys: human, simian and cross-immune types. Transplant. Proc. 4, 101.
Acquisition of human blood group antigens by Schistosoma mansoni 0. L. GOLDRING, *J. A. CLEGG, S. R. SMITHERS & R. J. TERRY National Institute for Medical Research, Mill Hill, London and Brunel University, Uxbridge, Middlesex (Received 1 March 1976) SUMMARY
Juvenile forms of Schistosoma mansoni (schistosomula) have been cultured in human blood of various specificities and tested for the presence of blood group substances on their surfaces. The tests employed were survival following transfer into rhesus monkeys immunized against human blood substances, mixed agglutination reactions, and immunofluorescence. A, B, H and Lewisb+ antigens were expressed at the surface when the parasites were cultured in blood of appropriate specificities. Rhesus, M N S, and Duffy antigens could not be detected on the parasite surface following culture. The evidence suggests that the expressed blood group antigens are of host origin and are acquired by the parasite during culture, probably in the form of glycolipids or megaloglycolipids. It is likely that these substances are also acquired by parasites in the bloodstream of man. They may serve to mask surface parasite antigens, and so enable schistosomes to evade parasitespecific humoral or cellular immune responses. INTRODUCTION A close and intimate association between host-like antigenic determinants and the surface of the trematode parasite S. mansoni was first demonstrated by Smithers, Terry & Hockley (1969). Schistosomes grown to maturity in mice survived well on transfer into the portal systems of rhesus monkeys, but were rapidly destroyed if these monkeys had first been immunized against mouse erythrocytes. The schistosomes were killed by antibodies directed against sites on the parasite surface which presumably shared antigen specificity with mouse erythrocytes. The existence of this close association of host-like antigenic determinants and schistosome surfaces has now been amply confirmed (Clegg, Smithers & Terry, 1970; 1971a and b; Cioli and Neis (1972); Perez & Terry, 1973; McLaren, Clegg & Smithers, 1975). It has been suggested that these determinants are truly of host origin, and that their acquisition and disposition at the parasite surface enables the adult parasite to evade the efferent arm of the host immune response (Clegg, 1974; Terry & Smithers, 1975). The evolution of such a protective device might explain the long survival of adult schistosomes in the blood of experimental animals and man. An understanding of the nature and mode of acquisition of host antigens by schistosomes might well be essential for any rational progress towards the development of vaccines against thesef important parasites. Clegg, Smithers & Terry (1971b) grew juvenile schistosomes (schistosomula) in culture media containing human blood of various specificities, and transferred them into the portal systems of monkeys immunized against human erythrocytes. These preliminary experiments suggested that the schistosomes acquired antigens during culture, some of which showed A or B blood group specificity. The aims of Present address: Wellcome Research Laboratories, Beckenham, Kent. Correspondence: Dr S. R. Smithers, National Institute for Medical Research, Mill Hill, London N.W.7. *
181
0. L. Goldring et al.
182
the present investigation were to identify exactly which human blood group antigens were acquired during culture and to determine in what form they were acquired.
MATERIALS AND METHODS Parasite. A Puerto Rican strain of S. mansoni was used throughout this work; its maintenance has been described previously (Smithers & Terry, 1965). Human blood. Four human bloods were used in this investigation; blood group antigen specificities are shown in Table 1. TABLE 1. Blood group specificities of human bloods used for the cultivation of
schistosomula ABO
C
A1 B 0 A1B
-
+
+
+
-
+
-
+ +
+ +
-
+
+ +
+ +
+ -
-
-
-
+
+
+
-
D
E
c
e
M
N
Fya
Leb
+
+
+
+ +
+ -
+ +
+
-
+
S
Human blood group substances. Purified human A blood group glycoprotein was a gift from Professor Winifred Watkins (Lister Institute of Preventive Medicine). Crude A, blood group 'megalolipid' was prepared from A1 cells by the methods of Gardas & Koscielak (1973 and 1974) and lyophilised. Monospecific blood group antisera. All sera were inactivated at 560C for 30 min. The following were obtained commercially from Ortho Pharmaceuticals: human anti A; B; rhesus C, c, D, E, and e; Duffy Fya+; S; rabbit anti-M; N; and Lewisb+. Other sera employed were a rabbit anti-blood group H substance, absorbed with human AIB erythrocytes (gift of Professor Watkins); and a rabbit anti-human globulin (Gift of Dr K. L. G. Goldsmith, MRC Blood Group Reference Laboratory). Normal rabbit serum absorbed with A1B erythrocytes provided the control serum. Fluorescein-labelled antisera. The following were obtained from Miles Laboratories: IgG fraction of FITC-labelled rabbit anti-human IgG; IgG fraction of FITC-labelled rabbit anti-human IgM; IgG fraction of FITC-labelled goat anti-rabbit IgG. Immunization ofmonkeys. Rhesus monkeys weighing between 2-6 and 4.5 Kg were each immunized with 0 3 mg of purified A glycoprotein emulsified in Freund's complete adjuvant (FCA). Four weeks later booster inoculations were given using the same immunization dose but without adjuvant. Inoculations were given both s.c. and i.m. in several sites. Cultivation ofschistosomula in human blood. Schistosomula were collected by allowing cercariae to penetrate isolated mouse skin and cultured in vitro for periods of up to 15 days, according to the methods of Clegg & Smithers (1972). The culture medium consisted of equal volumes of fresh human or monkey serum, and Earle's balanced salt solution with 0 5% lactalbumin hydrolysate and antibiotics (ELac). To this were added human or monkey erythrocytes, at a final concentration of 1%, or blood group substances at the concentrations specified in the descriptions of the experiments. The medium was gassed with 8% CO2 in air and the pH maintained at 7-4. The schistosomula were cultured in Leighton tubes, each containing 200-250 schistosomula at 37°C. The medium was changed every 3-4 days but the original erythrocyte inocula were retained. Satisfactory growth was judged by the criteria of Clegg (1965) which were established from in vivo observations. Transfer ofschistosomula to monkeys. Schistosomula for transfer were washed in ELac containing 1%/ normal monkey serum and then transferred into the portal systems of normal and immunized monkeys by cannulation of the mesenteric veins (Smithers & Terry, 1967). Five weeks later, when surviving worms had reached maturity, the monkeys were killed, perfused and the adult worms recovered (Smithers & Terry, 1965). Mixed agglutination. The principle of this test (Coombs, Bedford & Rouillard, 1956) is the formation of mixed aggregates of two cell types by antibody reacting with similar antigenic determinants occurring on the surfaces of the different cells. Fifteen-day schistosomula were removed from culture and washed several times with ELac containing 1% BSA (ELacBSA). They were allowed to settle, the washing fluid was removed and replaced by 50 ul of the appropriate antiserum. The schistosomula were incubated at 20°C for 1 hr, washed three times with ELac-BSA and then placed in 041 ml of a 2% erythrocyte suspension in ELac-BSA. The preparation, containing 10-30 schistosomula, was transferred to a microscope slide and examined for mixed agglutination with a Leitz inverted microscope. Positive and negative reactions are shown in Plates la and lb respectively. Immunofluorescence. The indirect method was employed. Schistosomula were well washed with ELac-BSA, and after
Human blood group antigens and S. mansoni
183
concentration 50 pl of the appropriate antiserum was added. They were incubated at room temperature for 30 min and then washed three times with ELac-BSA. 50 p1 of the appropriate undiluted FITC-antibody was added and the schistosomula incubated for a further 30 min at 20'C. The schistosomula were washed four times in ELac-BSA, transferred to a microscope slide and examined for fluorescence with a Reichert Binolux microscope; Wotan HB 200 W mercury vapour lamp; BG 12/5-2 mm exciter and Ilford 107 barrier filters.
RESULTS (a) In vivo experiments: expression of A substance Six rhesus monkeys were each immunized with purified A glycoprotein in FCA and four monkeys were inoculated with FCA alone. Booster inoculations were given 30 days later. Schistosomula were cultured for 15 days in either A 'secretor' blood, A 'non-secretor' blood or B 'secretor' blood. One week after the booster inoculation, these schistosomula were surgically transferred into the mesenteric veins of immunized and control monkeys as shown in Table 2. The numbers transferred varied between 100 and 150, but were accurately known for each transfer. Care was taken TABLE 2. Survival of S. mansoni in monkeys immunized against blood group substance A
a
Monkey
Immunization
Blood culture
Percentage of worm recovery
700 701 706 707 702 705 708 709 703 704
Blood group Substance A+FCA FCA FCA Blood group Substance A+FCA FCA FCA Blood group Substance A+FCA
A secretor A secretor A secretor A secretor A non-secretor A non-secretor A non-secretor A non-secretor B secretor B secretor
1 3 81 79 5 0 48 69 80 79
b
__ _ ~~~~~~~~~~~.."...................... t ~ .+ !. Am-| As
FIG. 1. (a) Positive mixed agglutination between 15-day schistosomulum (anterior end) and erythrocytes (Magnification x 720); (b), negative mixed agglutination (Magnification x 600).
"
184
0. L. Goldring et al.
to ensure that only fully viable and well-developed schistosomula were transferred. Five weeks later, the monkeys were killed, and the adult schistosomes were recovered by perfusion and counted. The results summarized in Table 2 are very clear. Recoveries from control monkeys, inoculated with FCA alone are high, validating the transfer and recovery procedures. Schistosomula cultured in A blood, either 'secretor' or 'non-secretor' were almost totally destroyed on transfer into anti-A monkeys, whereas those cultured in B 'secretor' blood survived normally. It is inferred that the schistosomula grown in A group blood, irrespective of secretor status, had A antigens on their surfaces. Those schistosomula which were grown in B blood did not possess A antigen. It is therefore suggested that those schistosomula which possessed A antigens had acquired them from the blood in which they had been grown.
(b) In vitro experiments The foregoing experiment had suggested the acquisition of A antigens by schistosomula grown in A blood. Unfortunately experiments of this type could not be used to study the acquisition of B and H blood group antigens as rhesus monkeys are known to possess B and H type substances in their saliva, although not on their erythrocytes, and so could not be immunized against them (Wiener, MoorJankowski & Socha, 1972). The possible acquisition of these antigens had perforce to be studied by in vitro methods using mixed agglutination and fluorescent antibodies. (1) Expression of blood group substances A, B and H as determined by mixed agglutination reactions. Schistosomula were cultured in human bloods of specificities A, B, 0 and AB for 15 days. At the end of this period, the schistosomula were reacted with antisera to blood group substances A, B and H and the indicator erythrocytes, and examined for mixed agglutination. Table 3 summarizes the design of this experiment and the results obtained. Positive reactions were seen where the blood used in culture and the indicator erythrocytes shared the same blood group specificity, and where the antiserum was directed against that specificity. Thus, schistosomula expressed only A antigen when cultured in A blood; only B antigen when cultured in B blood; only H antigen when cultued in 0 blood; and both A and B antigens when cultured in AB blood. (2) Expression of blood group substances A and B when cultured in serum. This experiment was designed to determine whether schistosomula acquire the blood group antigens directly from erythrocytes, or whether they can acquire them from serum. Glycoproteins with A B H specificity are found in the serum and other body fluids of 'secretors' but not 'non-secretors'; glycolipids with this specificity may occur in the sera of both (Gardas & Koscielak, 1973). Accordingly, schistosomula were cultured for 15 days in B serum, A 'secretor' serum or A 'nonsecretor' serum. The various sera were cleared from possible contamination with erythrocyte ghosts by centrifugation at 11,000 g for 30 mins. As schistosomula require erythrocytes for normal development O cells were added to all cultures. The schistosomula were then tested for the acquisition of A and B antigens by mixed agglutination. The plan and results of this experiment are summarized in Table 4. Schistosomula acquired B antigen when grown in B serum, and A antigen when grown in A serum, irrespective of secretor status. Seemingly schistosomula can acquire blood group antigens of A B H type from serum, and these antigens are likely to be in the form of glycolipids. (3) Acquisition of A blood group substance from an A 'megaloglycolipid' preparation. Gardas & Koscielak (1973 and 1974) have described water-soluble antigens of the human erythrocyte membrane with A, B, H and I activity. Chemical evidence suggests that these antigens are complex glycolipids, containing 20-40 sugar residues per mole of ceramide; these glycolipids have been designated 'megaloglycolipids'. Since we had some indirect evidence that schistosomula might be acquiring glycolipids from serum, we investigated their capacity to take up megalolipids prepared from A erythrocytes. Schistosomula were cultured in either B blood, or B blood to which had been added A megaloglycolipid at a concentration of 90 pg/ml. They were then examined for mixed agglutination with the appropriate antisera and indicator cells. All schistosomula were positive when tested with anti-B serum and B erythrocytes, and additionally, those cultured in the presence of A megaloglycolipid were positive
185
Human blood group antigens and S. mansoni TABLE 3. Detection of A, B and H antigens on schistosomula by mixed agglutination
Blood in culture
Antiserum
Indicator erythrocytes
No. of schistosomula observed
A B 0 A B 0 A B 0 A B 0 A B 0 A B 0 A B 0 A B 0
10 8 9 11 8 7 9 7 10 9 8 6 9 8 7 8 11 11 11 10 10 10 14 8
anti-A anti-B anti-H
A
B
*NRS anti-A anti-B anti-H
0
NRS anti-A anti-B anti-H
AB
NRS anti-A anti-B anti-H NRS
Percentage showing mixed agglutination 100 0 0 0 0 0 0 100 0 0 0 0 0 0 100 0 0 0 100 100 0 0 0 0
The 0 indicator erythrocytes were Lea /Leb- in order to eliminate possible reactions of Lewis antigens with the rabbit anti-H serum. * Normal rabbit serum absorbed with A1B erythrocytes.
TABLE 4. Detection of A and B antigens on schistosomula cultured in A and B serum
Culture Serum
Cells
B Secretor
0
A Secretor
0
A
0
Non-secretor
Antiserum
Indicator erythrocytes
No. of schistosomula observed
Anti-A Anti-B -
A B A B
14 8 9 12
0 100 0 0
Anti-A Anti-B
A B A B A B
12 11 7 8 14 11
100 0 0 0 100 0
Anti-A Anti-B
Percentage showing mixed agglutination
186
0. L. Goldring et al.
when tested with anti-A serum and A cells. Evidently schistosomula can acquire blood group antigens which are in megaloglycolipid form. (4) Failure to detect expression of M and N antigens by mixed agglutination. M and N antigens may be detected by direct erythrocyte agglutination with rabbit anti-M and anti-N antisera, containing specific IgM antibodies. Accordingly, schistosomula were cultured for 15 days in B type blood where the cells were M and N positive. They were then tested for their capacity to produce mixed agglutination reactions with M and N positive cells in the presence of rabbit anti-M and anti-N antisera. No positive reactions were observed, indicating that schistosomula do not acquire M and N antigens in culture. (5) Investigations on the expression of blood group antigens using immunofluorescence. Schistosomula were grown for 15 days in a range of human bloods, various members of which were positive for the A, B, S, rhesus (C.D.E.c.e), Lewis (Leb)+ and Duffy (Fy')+ blood group antigens (Table 1). The schistosomula were reacted with the relevant blood grouping sera, then treated with FITC-labelled antisera to human and rabbit immunoglobulins as appropriate. The usual controls were included. Strong positive specific immunofluorescence was seen on testing for A and B antigens, and weak reactions for the Lewis antigen. Tests for all other antigens were negative. We did not use antigenpositive red cells as positive immunofluorescence controls because of reported difficulties associated with the quenching effect of haemoglobin (Nairn, 1969). The activity of all serological reagents was however demonstrated by antiglobulin (Coombs) tests on antigen-positive red cells. The immunofluorescence studies therefore provide additional evidence that A and B antigens can be acquired by schistosomula in culture; there is evidence that Lewis`+ antigen may be acquired, but no evidence that the S, rhesus and Duffy antigens are acquired. DISCUSSION These experiments provide clear evidence for the presence of A, B and H substances on the surfaces of schistosomes cultured in human blood. The presence of a particular specificity on the parasite surface depends on that specificity being present in the blood used for culture. The simplest view is that the A, B and H substances are of host origin and are acquired by the parasite, but other explanations are not necessarily excluded by the data. It is perhaps surprising that the schistosomes seemed to acquire H antigen only when they were grown in 0 blood, as H antigen is also present on A, B and AB erythrocytes. It is known, however, that the amount of H antigen is less on these cells than on 0 cells, and it is possible that the more abundant A and B antigens compete strongly for sites on the parasite surface. Regarding other blood group antigens, there is preliminary evidence for the acquisition of Lewis (Leb+) antigen, but no evidence for the acquisition of M, N,S, rhesus and Duffy antigens. Our experiments indicated that schistosomes could acquire A and B antigens from the sera of A and B positive donors, in the absence of A and B erythrocytes. This occurred irrespective of the secretor status of the donor. Non-secretors do not possess the soluble glycoprotein A B H antigens in their body fluids, so that it seems unlikely that the antigens acquired by the parasites from serum was in the form of glycoproteins. High-speed centrifugation of the sera used for culture removed erythrocytes and ghosts. Nevertheless, it is possible that some membrane fragments remained in the sera, and that these provided the source of antigens. An alternative view, is that A B H substances may occur in the plasma of both secretors and non-secretors in the form of the megaloglycolipids described by Gardas & Koscielak (1973 and 1974). These workers showed that the glycolipids had the unique property of combining with cells and conferring upon them appropriate surface antigenic specificity. Further, they believe that these megaloglycolipids, present in the erythrocyte membrane, might be originally derived from the blood plasma. Our experiments have shown that culture in A megaloglycolipid conferred A specificity on the schistosome surface. In the absence of contrary evidence, it seems not unreasonable to believe that the A B H blood group specific substances present at the surface of schistosomes cultured in human blood, are derived from the plasma, and that antigens are similarly acquired in the bloodstream of man. Some support for this view is provided by the studies of other blood group antigens. Lewis substance
Human blood group antigens and S. mansoni
187
is acquired by schistosomes, and this is known to exist as a glycolipid in plasma and to have the property of attaching to cells (Marcus & Cass, 1969). The antigens of other groups, M, N, S, rhesus and Duffy, are believed to be glycoproteins, confined to the erythrocyte membrane, and these are not acquired by the parasite. These investigations provide no direct support for the view that the acquisition of host antigens enables the schistosome to evade host immunity (Clegg, 1974; Terry & Smithers, 1975). But it is difficult to escape the conclusion that the acquisition of substantial amounts of host blood group substances will serve to mask target antigens on the parasite surface and thus reduce the effectiveness of specific humoral or cellular immunity. We are grateful to Miss Elaine Holder and Mr K. Broomfield for their skilled technical assistance.
REFERENCES CIOLI, D. & NEIS, R. (1972) Antigeni dell'ospite in Schistosoma mansoni. Parassitologia, 14, 73. CLEGG, J.A. (1965) In vitro cultivation of Schistosoma mansoni. Exp. Parasit. 16, 133. CLEGG, J.A. (1974) Host antigens and the immune response in schistosomiasis. Parasites in the immunized host: mechanisms of survival, CIBA Foundation Symposium, 25 (new series), 161. CLEGG, J.A. & SMiTnERs, S.R. (1972) The effects of immune rhesus monkey serum on schistosomula of Schistosoma mansoni during cultivation in vitro. Int. J. Parasit. 2, 79. CLEGG, J.A., SMITHERS, S.R. & TERRY, R.J. (1970) 'Host' antigens associated with schistosomes: observations on their attachment and their nature. Parasitology, 61, 87. CLEGG, J.A., SMITHERS, S.R. & TERRY, R.J. (1971a) Concomitant immunity and host antigens associated with schistosomiasis. Int. J. Parasit. 1, 43. CLEGG, J.A., SMITHERS, S.R. & TERRY, R.J. (1971b) Acquisition of human antigens by Schistosoma mansoni during cultivation in vitro. Nature (Lond.), 232, 653. COOMBS, R.R.A., BEDFORD, D. & ROUILLARD, L.M. (1956) A and B blood group antigens on human epidermal cells demonstrated by mixed agglutination. Lancet, i, 461. GARDAS, A. & KoscIELAK, J. (1973) New form of A-, Band H-blood group active substances extracted from erythrocyte membranes. Europ. J. Biochem. 32, 178. GARDAS, A. & KOSCIELAK, J. (1974) Megaloglycolipidsunusually complex glycosphingolipids of human erythrocyte membranes with A, B, H and I blood group specificity. FEBS letters, 42, 101.
McLAREN, D.J., CLEGG, J.A. & SMITHERS, S.R. (1975) Acquisition of host antigens by young Schistosoma mansoni in mice: correlation with failure to bind antibody in vitro. Parasitology, 70, 67. MARCUS, D.M. & CASS, L.E. (1969) Glycosphingolipids with Lewis bloodgroup activity; uptake by human erythrocytes. Science, 164, 553. NAIRN, R.C. (1969) Fluorescent protein tracing, 3rd edn (Ed. by R.C. Nairn), E. & S. Livingstone Ltd. Edinburgh and London. PEREZ, H. & TERRY, R.J. (1973) The killing of adult Schistosoma mansoni in vitro in the presence of antisera to host antigenic determinants and peritoneal cells. Int. 5. Parasit. 3, 499. SMITHERS, S.R. & TERRY, R.J. (1965) The infection of laboratory hosts with cercariae of Schistosoma mansoni and the recovery of the adult worms. Parasitology, 55, 695. SMITHERS, S.R. & TERRY, R.J. (1967) Resistance to experimental infections with Schistosoma mansoni in rhesus monkeys induced by the transfer of adult worms. Trans. roy. Soc. trop. Med. Hyg. 61, 517. SMITHERS, S.R., TERRY, R.J. & HOCKLEY, D.J. (1969) Host antigens in schistosomiasis. Proc. roy Soc. B. 171, 483. TERRY, R.J. & SMITHERS, S.R. (1975) Evasion ofthe immune response by parasites. Symposia Soc. exp. Biol. 29, 453. WEINER, A.S., MOOR-JANKOWSKI, J. & SOCHA, W.W. (1972) Principles of blood grouping in apes and monkeys: human, simian and cross-immune types. Transplant. Proc. 4, 101.
