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Demonstration of a homologous anaphylactic antibody in rabbits infected with Schistosoma mansoni
EXPERIMENTAL
20, 278-287
PARASITOLOGY
Demonstration in
of
Rabbits
(1967)
a Homologous
Infected
with N.
Georgetown E. H. Sadun,
of
and
M. J. Schoenbechler
Research, Washington,
for publication,
E. H.,
mansonil
Washington, D.C. 20007
E. L. Becker,
(Submitted N. J., SADUN,
Schistosoma
Antibody
J. Zvaifler
University,
Walter Reed Army Institute
ZVAIFLER,
Anaphylactic
BECKER,
21 February
E. L.,
D.C. 20012
1967)
AND SCHOENBECHLER,
M.
J.
1967.
Demonstration of a homologous anaphylactic antibody in rabbits infected with Schistosoma mansoni. Experimental Parasitology 20, 278-287. Groups of rabbits were exposed to 1000, 5000, or 25,000 Schistosoma mansoni cercariae. Flocculating antibodies were detected by the use of somatic antigens from eggs and cercariae, and by metabolic antigens from adult worms. Flocculating antibodies appeared in all the animals by the third week and persisted throughout the experiment. Highest titers were obtained with the adult metabolic antigen. Nine of eleven rabbits developed an antibody which produced passive cutaneous anaphylaxis in normal rabbits challenged with cercarial antigen. This antibody appeared 7-12 weeks following infection. It remained elevated for 3-6 weeks, often in high titers, and then either could no longer be demonstrated, or was detected at very low titers. Reinfection with 5000 cercariae at 36 weeks elicited an abrupt return of anaphylactic antibodies in half of the animals. The anaphylactic antibody was inactivated by heating at 56°C and by mercaptoethanol. No obvious correlation was observed between antibodies detected by slide flocculation tests and those detected by passive cutaneous anaphylaxis.
The serology and immunodiagnosis of schistosomiasis have been investigated for many years. The variety of diagnostic tests available is not surprising when one considers the antigenic complexity of the parasite and the various stages in its life cycle. A major deficiency of these tests has been that the antibodies which they detect appear to bear little relationship to the course or clinical features of the disease, or to the immunity which is acquired as a result of infection with schistosomes (Kagan and Pellegrino, 1961).
Investigations in recent years have pointed out the heterogeneity of the immune response. In addition to conventional gamma G and gamma M antibodies, another class of antibodies has been recognized which is characterized by its ability to fix to the tissues of the homologous species. These have been designated by a variety of terms: reaginic antibody, homologous anaphylactic antibody, or homocytotropic antibody. In general, anaphylactic antibody migrates in electrophoresis faster than gamma G antibody, and is inactivated by heating at 56°C or when treated with 2-mercaptoethanol. When introduced into the skin it persists at the injection site for many days or weeks, in contrast to gamma
‘Supported in part by funds from U.S. Army Medical Research and Development Command No. DA 49-193-MD2911, and the U.S. Public Health Service Al -07444. 278
ANTIBODY
DEMONSTRATION
G globulin which disappears in l-2 days. It is this property of skin fixation which provides the most useful measure of anaphylactic antibody. Zvaifler and Becker (1966) demonstrated that rabbits immunized with soluble protein and hapten antigens form a homologous anaphylactic antibody. This antibody gives passive cutaneous anaphylaxis in rabbit skin, persists at skin sites for long periods of time, is heat labile, is inactivated by treatment with mercaptoethanol, is nonprecipitating, and does not fix complement. The anaphylactic antibody is demonstrable 6-7 days after immunization and usually disappears by the third week. The occurrence of reagin-like antibodies in rats infected with the nematode, Nippostrongylus braziliensis was reported recently. Immunization of rats with freshly homogenized adult worms, with or without Freund’s adjuvant, did not induce the formation of anaphylactic antibody. Ogilvie (1964) suggested that reaginic antibody was stimulated only by a living organism, and that this type of antibody might be responsible for immunity to helminths. Later she reported similar results in rats and monkeys infected with S. mansoni (Ogilvie et al., 1966). These findings led us to a series of investigations to determine whether rabbits infected with schistosomes develop anaphylactic antibody, whether the antibodies produced relate to any clinical features of the disease, and finally how the anaphylactic antibody correlates with the flocculating antibodies which are known to develop during the course of this infection. A preliminary account of this work has been presented elsewhere (Zvaifler et al., 1966). MATERIALS
AND METHODS
Animals
Albino rabbits weighing 2500-3000 gm were used for passive cutaneous anaphy-
IN INFECTED
RABBITS
279
laxis reactions and for the schistosome infections. All animals were fed a standard diet. They were bled from the central ear vein prior to infection and at regular intervals throughout the experiment. The principles of laboratory animal care as promulgated by the National Society for Medical Research were observed. Experimental Infections
Twelve rabbits were divided into three groups of four animals each, and exposed percutaneously to either 1000, 5000, or 25,000 cercariae. Thirty-six weeks after the initial exposure, all of the 11 surviving animals were reexposed percutaneously with 5000 cercariae. Serologic Tests
The slide flocculation tests were performed according to the method of Anderson ( 1960). Eggs, cercariae, and adult schistosome excretions and secretions (adult ES) were employed as antigens. The preparation and adsorption onto cholesterol lecithin crystals was the same as has been described previously (Sadun et al., 1965). Passive Cutaneous Anaphylaxis (PCA)
Intradermal injections of 0.2 ml of whole rabbit serum or serum dilutions in phosphate buffered saline (pH 7.2) were made into the backs of freshly shaven albino rabbits. All tests were performed in duplicate. After a latent period of approximately 72 hours, the animals were challenged intravenously with 1.0 ml of a lyophilized preparation of schistosome cercariae containing approximately 27 mg protein/ml and 1.0 ml of 5 % pontamine sky blue dye in 0.15 M saline. In a positive test, a blue spot develops at the intradermal injection site; the size and intensity of the blueing is roughly proportional to the amount of homologous anaphylactic antibody present. The result-
280
ZVAIFLER,
SADUN, BECKER,
ing lesions were measured and recorded after 30-60 minutes. The reciprocal of the highest dilution giving a lesion of 5 x 5 mm or greater was taken as the passive cutaneous antibody titer. RESULTS
Flocculation Tests with Sera from Experimentally Infected Rabbits Sera taken at weekly intervals from each of the infected rabbits were examined in the slide flocculation test against cercarial, egg, and adult ES antigens. The results obtained with the cercarial antigen are shown in Fig. 1. The median titer of the serum from each of three groups of animals infected with 1000, 5000, and 25,000 cercariae, respectively, is plotted throughout the course of the experiment. Flocculating antibody was demonstrable 3-4 weeks following infection, reached a peak by weeks 8-10,
AND SCHOENBECHLER
and then declined gradually but remained elevated throughout the study. There was very little change in the median titers following reinfection on week 36. In general, the animals infected with 25,000 cercariae, had higher titers than those exposed to 1000 or 5000 cercariae. The results with the egg antigen were very similar to those with cercarial antigen in appearance time, overall median titers and lack of an anamnestic response following reinfection. Figure 2 shows the results obtained using adult excretions and secretions as antigen. The median titers were higher than those seen with egg or cercarial antigens, especially in the rabbits infected with 25,000 cercariae. The overall pattern was similar to that seen with the other flocculating antibodies, with appearance of antibody by week 3, and persistence of antibody throughout the study. There was a modest antibody rise 6-8 weeks after reinfection. After week
TITER 2046 1024 512 256 126 64 32 16
6 4 2
5
IO
I5
20
25
30
35
40
45
WEEKS
FIG. 1. The median slide flocculation titers with cercarial fection with 1000 (-), 5000 (. . . ), and 25,000 (---) 5,000 cercariae on week 36 is designated by the arrow.
antigen obtained in rabbits following incercariae of S. mansoni. Reinfection with
ANTIBODY
DEMONSTRATION
IN
INFECTED
281
RABBITS
TITER 20461024 512
-
256
-
126 64 32 16 642I
I 5
I IO
I I5
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I
25
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30
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35
I
40
I
45
WEEKS FIG. 2. The median slide flocculation titers with adult metabolic products as antigen obtained in rabbits following infection with 1000 (-), 5000 (. . .) and 25,000 (---) cercariae of S. munsoni. Reinfection with 5000 cercariae on week 36 is designated by the arrow.
5, flocculating antibody was demonstrated in the serum from every infected animal with each of the three antigens. PCA Reactions with Sera from Experimentally Infected Animals
The individual PCA titers for the three rabbits infected with 1000 cercariae are shown in Fig. 3. No PCA antibody was detected before week 6, then it appeared abruptly, remained elevated for lo-12 weeks and thereafter was no longer demonstrable. Reinfection on week 36 resulted in an abrupt secondary response in one animal, a delayed secondary response in another, and no response in the third. There was no correlation of PCA titers and flocculating antibody titers. Rabbit No. 1472, the animal which showed both a primary and an abrupt secondary response, was the weakest reactor in the slide flocculation test with all three antigens. The results obtained with the 4 rabbits
infected with 5000 cercariae are shown in Fig. 4. Three developed PCA antibody beginning at week 6 and reached a peak titer by week 9-13. Two of the three animals showed a secondary response equal to, or greater than, the original PCA titer following reinfection at week 36. The anaphylactic antibody response of the rabbits infected with 25,000 cercariae is depicted in Fig. 5. Three of the four animals had demonstrable PCA activity; in two the primary response was biphasic, with an initial short-lived appearance of antibody about week 6, followed by a more prolonged elevation from weeks 12-19. Following reinfection in all three animals which showed a primary response, there was a prompt reappearance of anaphylactic antibody. The one rabbit which did not show a primary response did not demonstrate PCA activity after reinfection. The patterns of anaphylactic antibodies in the three groups of animals are compared
ZVAIFLER,
282
SADUN, BECKER, 1000
500
AND SCHOENBECHLER CERCARIAE
400 300
45.j """""". ,472 ..-... 1477-
200
PCA TITER
loo 90 80
70 60 50 40
WEEKS
FIG. 3. The individual titers of homologous passive cutaneous anaphylactic antibody obtained 3 rabbits following infection with 1000 cercariae of S. mansoni. Reinfection with 5000 cercariae week 36 is designated by the arrow.
5000
in on
CERCARIAE ,606 -..-..,679
,Y"""."""""
1696 ,699
."""""""".
PCA TITER
0
4
8
12
16
20
24
26
32
36
40
44
46
52
56
60
WEEKS FIG. 4. The individual titers of homologous passive cutaneous anaphylactic antibody obtained 4 rabbits following infection with 5000 cercariae of S. mansoni. Reinfection with 5000 cercariae week 36 is designated by the arrow.
in on
ANTIBODY
DEMONSTRATION 25,000
IN INFECTED
RABBITS
283
CERCARIAE
1700 .-..I ,701 ~~~~~~~. 1702 --170 PCA TITER
0
4
6
I2
16
20
24
,26
32
36
40
44
46
62
56
60
WEEKS
FIG. 5. The individual titers of homologous passive cutaneous anaphylactic rabbits following infection with 25,000 cercariae of s. munsoni. Reinfection week 36 is designated by the arrow.
in Fig. 6. Antibody activity was seldom present before week 6, following the initial infection. In general, the group infected with 1000 cercariae showed an abrupt rise to the highest PCA titers which then fell rapidly. The animals infected with 25,000 cercariae had the lowest titers, which remained elevated for the longest period of time. The response of the group infected with 5000 cercariae was intermediate. The PCA titers in all three groups declined by week 20. With reinfection there was a more abrupt appearance of antibody, which in most cases remained elevated throughout the next 20 weeks. The secondary responses of the rabbits infected with 5000 and 25,000 cercariae were equal to, or greater than, the initial response. In the group infected with 1000 cercariae, the secondary response was less evident than the primary one. Nine of the eleven infected rabbits demonstrated anaphylactic antibody. The two animals which did not show a primary
antibody obtained in 4 with 5000 cercariae on
response did not develop anaphylactic antibody following reinfection. Two additional animals which had anaphylactic activity after the initial infection did not show a secondary response after reinfection. There was no correlation of the two anaphylactic antibody titers with the flocculating antibody titers. Susceptibility of Flocculating and Anaphylactic Antibody to Heat and Mercaptoethanol Treatment
Heating serum at 56°C for 60-120 minutes resulted in a reduction of anaphylactic acitivty by 50-75 % . Anaphylactic activity was completely destroyed by heating beyond 2 hours. Similar treatment had no effect on flocculating antibody titers. Exposure of serum to 0.1 M 2-mercaptoethanol followed by alkylation with an excess of 0.02 M iodoacetamide resulted in complete loss of anaphylactic activity, but there was no effect on flocculating: antibodv.s _._ _.---
284
ZVAIFLER,
xo4mm-
SADUN,
BECKER,
25.000
AND
SCHOENBECHLER
CERCARIAE
ITO2 _1_ 110 -
mPCA TITFR
WEEKS
FIG. 6. A composite showing the patterns of development of homologous anaphylactic antibody in rabbits infected with 1000, 5000 and 25,000 cercariae of S. mansani, and when reinfected with 5000 cercariae on week 36.
ANTIBODY
DEMONSTRATION
DISCUSSION
The results of these studies indicate that there are many features distinguishing the flocculating from the anaphylactic antibodies which develop in rabbits with schistosomiasis. All of the animals produced flocculating antibodies which appeared by weeks 3-4, and persisted throughout the study. Anaphylactic antibody was present by weeks 6-8 in nine of eleven infected rabbits; it persisted for several weeks in some, but had declined by week 20 in all. Although there was a direct correlation between the number of infecting organisms and the height of flocculating antibody titers, no such correlation was noted for anaphylactic antibody. When the rabbits were reinfected on week 36, there was very little increase in flocculating antibody titers, but seven of eleven rabbits showed a secondary anaphylactic antibody response. Flocculating antibodies are heat stable, not sensitive to mercaptoethanol and appear in the 7s gamma G globulin fraction on DEAE cellulose chromatography. Anaphylactic antibody is destroyed by heating, is inactivated by reduction and alkylation (Zvaifler and Becker, 1966), and is eluted from DEAE cellulose after the flocculating antibody (Zvaifler, unpublished abstract). Our results with relation to the time course of development of anaphylactic antibody in rabbits with schistosomiasis were remarkably similar to those found with rats (Ogilvie et al., 1966). Reaginic-type antibodies were found in the serum of rats 4 weeks after infection with S. mansoni. These persisted until week 12, and then disappeared. Sera taken one week after a second exposure were strongly positive. Monkeys showed reagins 6-16 weeks after exposure, and became negative within 4 weeks. All sera taken one week after reexposure were positive, and in some remained so for up to 20 weeks. Infection
IN
INFECTED
RABBITS
285
with irradiated cercariae resulted in negative or only weakly positive results even with repeated exposure (Ogilvie et al., 1966). Hsu and Hsu (1966) reported somewhat similar results in monkeys infected with S. japonicum. They found that 5 of 15 monkeys developed PCA positive sera; 2 in 38 days, 2 in 188 days and 1 in 592 days after the first inoculation. In contrast to Ogilvie’s findings they identified reagin-like antibody in the sera of 23 of 38 monkeys exposed to X-irradiated cercariae of S. japonicum. The antibodies did not appear in any of the animals before day 60 of infection, and in most they were not seen until many months or years, and only multiple reinfections. The situation in man and chimpanzees has so far been studied only in a limited number of specimens. Serum from individuals infected with S. mansoni or S. haematobium gave PCA reactions in the skin of rhesus monkeys (Sadun et al., 1966). The persistence of anaphylactic antibody in helminthic infections and the secondary response after reinfection differs from that which is seen when rabbits are immunized with soluble protein and haptene-protein antigens (Zvaifler and Becker, 1966). A primary immunization with soluble protein antigens in Freund’s complete or incomplete adjuvant is followed in 6-10 days by the appearance of low titers of anaphylactic antibody in one-half to two-thirds of the animals. The antibody rapidly disappears and is not recalled by subsequent immunization. A very similar situation has been reported in rats (Mota, 1964). The reason for the greater percentage of animals developing anaphylactic antibody, the higher PCA titers and the persistence of the antibody in animals with schistosomiasis is not known. The living antigen with its complex life cycle may result in a prolonged period of antigenic stimulation. The long latent period between exposure and the ap-
286
ZVAIFLER,
SADUN,
BECKER,
pearance of anaphylactic antibody may mean that the parasite does not become antigenic until it reaches adulthood, or that the antigen has arrived at some anatomic locus, such as the mesentery, favorable for anaphylactic antibody production. It is of interest that reaginic type antibodies are usually associated with diseases characterized by an eosinophilic response in the blood, i.e., allergy and helminthic infections, and eosinophilia becomes prominent in the organs of rabbits 30-40 days after infection by schistosomes (Koppisch, 1937). This is approximately the time when anaphylactic antibody appears in the serum. Recent studies have shown that passive cutaneous anaphylaxis is mediated by two different kinds of antibodies. In the first, serum from one species produces PCA reactions in the skin of another species, e.g., rabbit serum in guinea pig skin. This type of reaction, heterologous anaphylaxis or heterocytotropic anaphylaxis, is mediated by 7s gamma G globulin. This class of antibody is not affected by heating, is only mildly inactivated by reduction and alkylation, and is isolated in the “fall through” peak on DEAE-cellulose chromatograph (Zvaifler and Barbaro, 1967). Passive cutaneous antibody reactions are obtained within hours after the introduction of the antisera into the skin of the heterologous species, but are negative after a latent period of 48 hours, presumably because the antibody is unable to remain fixed to the skin sites. This is the type of anaphylactic antibody described by Ivey and Slanga (1965) who showed that serum from rabbits infected with Trichinella or Toxocara produced PCA reactions in guinea pig skin. The flocculating antibodies in schistosomiasis belong to this class of antibodies. The second kind of anaphylactic antibody gives passive cutaneous anaphylactic reactions in the skin of the species from which the antibody is derived, i.e.,
AND
SCHOENBECHLER
rabbit antibody in rabbit skin. This reaction is called homologous anaphylaxis or homocytotropic anaphylaxis, and is mediated by antibodies which differ from the conventional 7s immunoglobulins. Rabbit homologous anaphylactic antibody is heat labile, inactivated by reduction and alkylation, migrates as a beta globulin, and is eluted from DEAB-cellulose after the gamma G globulins. When placed in rabbit skin there is a latent period of 48-72 hours before PCA reactions occur, but the antibody remains fixed to the skin for many days or weeks (Zvaifler and Becker, 1966). Studies are in progress to determine whether the homologous anaphylactic antibody described in this report plays any role in the immunity which develops in rabbits infected with Schistosoma mansoni. ACKNOWLEDGMENTS The technical help of Mrs. Jennifer is gratefully acknowledged.
Robinson
REFERENCES ANDERSON, R. Schistosoma
I. 1960. Serologic diagnosis of mansoni infections. I. Development of a cercarial antigen slide flocculation test. American Journal of Tropical Medicine and Hygiene
9, 299-303.
Hsu, H. F., AND Hsu, S. Y. Lr 1966. Reagin-like antibody in rhesus monkeys immune to Schistosoma japonicum. Zeitschrift fur Tropenmedizin und Parasitologic 17, 166-176. IVEY, M. H., AND SLANGA, R. 1965. An evaluation
of passive cutaneous anaphylaxis reactions with Trichinella and Toxocara antibodyantigen systems. American Journal of Tropical Medicine and Hygiene 14, 1052-1056. KAGAN, I. G., AND PELLEGRINO, J. 1961. A critical review of immunological methods for the diagnosis of bilharziasis. Bulletin World Health Organization 25, 61 l-674. KOPPISCH, E. 1937. Studies on schistosomiasis mansoni in Puerto Rico. II. The pathological anatomy of experimental schistosomiasis mansoni in the rabbit and albino rat. Puerto
ANTIBODY Rico Journal of Public Medicine 13, l-59.
DEMONSTRATION
Health
and
Tropical
I. 1964. The mechanism of anaphylaxis. I. Production and biologic properties of mast cell sensitizing antibody. Immunology 7, 681699. OGILVIE, B. M. 1964. Reagin-like antibodies in animals immune to helminth parasites. Nature MOTA,
204, 91-92. OGILVIE,
B. M.,
SMITHERS,
S. R., AND TERRY,
R. J.
1966. Reagin-like antibodies in experimental infections of Schistosoma mansoni and the passive transfer of resistance. Nature 209, 1221-1223. SADUN,
E. H., SCHOENBECHLER,
M. 1965. Multiple
M.
J., AND BENTZ, response in Schis-
antibody infections: Antigenic constituents in eggs, cercariae and adult (excretions and secretions) determined by flocculation reactions, cross absorption and double tosoma
mansoni
IN
INFECTED
RABBITS
287
diffusion studies. American Journal of Tropical Medicine and Hygiene 14, 977-995. SADUN, E. H., VON LICHTENBERG, F., HICKMAN, R. L., BRUCE, 3. I., SMITH, I. H., AND SCHOENBECHLER, M. J. 1966. Schistosomiasis mansoni in the chimpanzee: Parasitologic, clinical, serologic, pathologic and radiologic observations. American Journal of Tropical and Hygiene 15, 496-506. J., AND BECKER, E. L. 1966. Rabbit anaphylactic antibody. Journal of Experimental Medicine 123, 935-950. ZVAIFLER, N. J., SADUN, E. H., AND BECKER, E. L. Medicine ZVAIFLER, N.
1966. Anaphylactic (reaginic) antibodies in helminthic infections. Clinical Research 45, 336 (abstr.). ZVAIFLER, N. I., AND BARBARO, J. 1967. Anaphylactic antibody in the primary response of rabbits. International Archives of Allergy and Applied
Immunology,
31, 465474.
20, 278-287
PARASITOLOGY
Demonstration in
of
Rabbits
(1967)
a Homologous
Infected
with N.
Georgetown E. H. Sadun,
of
and
M. J. Schoenbechler
Research, Washington,
for publication,
E. H.,
mansonil
Washington, D.C. 20007
E. L. Becker,
(Submitted N. J., SADUN,
Schistosoma
Antibody
J. Zvaifler
University,
Walter Reed Army Institute
ZVAIFLER,
Anaphylactic
BECKER,
21 February
E. L.,
D.C. 20012
1967)
AND SCHOENBECHLER,
M.
J.
1967.
Demonstration of a homologous anaphylactic antibody in rabbits infected with Schistosoma mansoni. Experimental Parasitology 20, 278-287. Groups of rabbits were exposed to 1000, 5000, or 25,000 Schistosoma mansoni cercariae. Flocculating antibodies were detected by the use of somatic antigens from eggs and cercariae, and by metabolic antigens from adult worms. Flocculating antibodies appeared in all the animals by the third week and persisted throughout the experiment. Highest titers were obtained with the adult metabolic antigen. Nine of eleven rabbits developed an antibody which produced passive cutaneous anaphylaxis in normal rabbits challenged with cercarial antigen. This antibody appeared 7-12 weeks following infection. It remained elevated for 3-6 weeks, often in high titers, and then either could no longer be demonstrated, or was detected at very low titers. Reinfection with 5000 cercariae at 36 weeks elicited an abrupt return of anaphylactic antibodies in half of the animals. The anaphylactic antibody was inactivated by heating at 56°C and by mercaptoethanol. No obvious correlation was observed between antibodies detected by slide flocculation tests and those detected by passive cutaneous anaphylaxis.
The serology and immunodiagnosis of schistosomiasis have been investigated for many years. The variety of diagnostic tests available is not surprising when one considers the antigenic complexity of the parasite and the various stages in its life cycle. A major deficiency of these tests has been that the antibodies which they detect appear to bear little relationship to the course or clinical features of the disease, or to the immunity which is acquired as a result of infection with schistosomes (Kagan and Pellegrino, 1961).
Investigations in recent years have pointed out the heterogeneity of the immune response. In addition to conventional gamma G and gamma M antibodies, another class of antibodies has been recognized which is characterized by its ability to fix to the tissues of the homologous species. These have been designated by a variety of terms: reaginic antibody, homologous anaphylactic antibody, or homocytotropic antibody. In general, anaphylactic antibody migrates in electrophoresis faster than gamma G antibody, and is inactivated by heating at 56°C or when treated with 2-mercaptoethanol. When introduced into the skin it persists at the injection site for many days or weeks, in contrast to gamma
‘Supported in part by funds from U.S. Army Medical Research and Development Command No. DA 49-193-MD2911, and the U.S. Public Health Service Al -07444. 278
ANTIBODY
DEMONSTRATION
G globulin which disappears in l-2 days. It is this property of skin fixation which provides the most useful measure of anaphylactic antibody. Zvaifler and Becker (1966) demonstrated that rabbits immunized with soluble protein and hapten antigens form a homologous anaphylactic antibody. This antibody gives passive cutaneous anaphylaxis in rabbit skin, persists at skin sites for long periods of time, is heat labile, is inactivated by treatment with mercaptoethanol, is nonprecipitating, and does not fix complement. The anaphylactic antibody is demonstrable 6-7 days after immunization and usually disappears by the third week. The occurrence of reagin-like antibodies in rats infected with the nematode, Nippostrongylus braziliensis was reported recently. Immunization of rats with freshly homogenized adult worms, with or without Freund’s adjuvant, did not induce the formation of anaphylactic antibody. Ogilvie (1964) suggested that reaginic antibody was stimulated only by a living organism, and that this type of antibody might be responsible for immunity to helminths. Later she reported similar results in rats and monkeys infected with S. mansoni (Ogilvie et al., 1966). These findings led us to a series of investigations to determine whether rabbits infected with schistosomes develop anaphylactic antibody, whether the antibodies produced relate to any clinical features of the disease, and finally how the anaphylactic antibody correlates with the flocculating antibodies which are known to develop during the course of this infection. A preliminary account of this work has been presented elsewhere (Zvaifler et al., 1966). MATERIALS
AND METHODS
Animals
Albino rabbits weighing 2500-3000 gm were used for passive cutaneous anaphy-
IN INFECTED
RABBITS
279
laxis reactions and for the schistosome infections. All animals were fed a standard diet. They were bled from the central ear vein prior to infection and at regular intervals throughout the experiment. The principles of laboratory animal care as promulgated by the National Society for Medical Research were observed. Experimental Infections
Twelve rabbits were divided into three groups of four animals each, and exposed percutaneously to either 1000, 5000, or 25,000 cercariae. Thirty-six weeks after the initial exposure, all of the 11 surviving animals were reexposed percutaneously with 5000 cercariae. Serologic Tests
The slide flocculation tests were performed according to the method of Anderson ( 1960). Eggs, cercariae, and adult schistosome excretions and secretions (adult ES) were employed as antigens. The preparation and adsorption onto cholesterol lecithin crystals was the same as has been described previously (Sadun et al., 1965). Passive Cutaneous Anaphylaxis (PCA)
Intradermal injections of 0.2 ml of whole rabbit serum or serum dilutions in phosphate buffered saline (pH 7.2) were made into the backs of freshly shaven albino rabbits. All tests were performed in duplicate. After a latent period of approximately 72 hours, the animals were challenged intravenously with 1.0 ml of a lyophilized preparation of schistosome cercariae containing approximately 27 mg protein/ml and 1.0 ml of 5 % pontamine sky blue dye in 0.15 M saline. In a positive test, a blue spot develops at the intradermal injection site; the size and intensity of the blueing is roughly proportional to the amount of homologous anaphylactic antibody present. The result-
280
ZVAIFLER,
SADUN, BECKER,
ing lesions were measured and recorded after 30-60 minutes. The reciprocal of the highest dilution giving a lesion of 5 x 5 mm or greater was taken as the passive cutaneous antibody titer. RESULTS
Flocculation Tests with Sera from Experimentally Infected Rabbits Sera taken at weekly intervals from each of the infected rabbits were examined in the slide flocculation test against cercarial, egg, and adult ES antigens. The results obtained with the cercarial antigen are shown in Fig. 1. The median titer of the serum from each of three groups of animals infected with 1000, 5000, and 25,000 cercariae, respectively, is plotted throughout the course of the experiment. Flocculating antibody was demonstrable 3-4 weeks following infection, reached a peak by weeks 8-10,
AND SCHOENBECHLER
and then declined gradually but remained elevated throughout the study. There was very little change in the median titers following reinfection on week 36. In general, the animals infected with 25,000 cercariae, had higher titers than those exposed to 1000 or 5000 cercariae. The results with the egg antigen were very similar to those with cercarial antigen in appearance time, overall median titers and lack of an anamnestic response following reinfection. Figure 2 shows the results obtained using adult excretions and secretions as antigen. The median titers were higher than those seen with egg or cercarial antigens, especially in the rabbits infected with 25,000 cercariae. The overall pattern was similar to that seen with the other flocculating antibodies, with appearance of antibody by week 3, and persistence of antibody throughout the study. There was a modest antibody rise 6-8 weeks after reinfection. After week
TITER 2046 1024 512 256 126 64 32 16
6 4 2
5
IO
I5
20
25
30
35
40
45
WEEKS
FIG. 1. The median slide flocculation titers with cercarial fection with 1000 (-), 5000 (. . . ), and 25,000 (---) 5,000 cercariae on week 36 is designated by the arrow.
antigen obtained in rabbits following incercariae of S. mansoni. Reinfection with
ANTIBODY
DEMONSTRATION
IN
INFECTED
281
RABBITS
TITER 20461024 512
-
256
-
126 64 32 16 642I
I 5
I IO
I I5
I
20
I
25
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45
WEEKS FIG. 2. The median slide flocculation titers with adult metabolic products as antigen obtained in rabbits following infection with 1000 (-), 5000 (. . .) and 25,000 (---) cercariae of S. munsoni. Reinfection with 5000 cercariae on week 36 is designated by the arrow.
5, flocculating antibody was demonstrated in the serum from every infected animal with each of the three antigens. PCA Reactions with Sera from Experimentally Infected Animals
The individual PCA titers for the three rabbits infected with 1000 cercariae are shown in Fig. 3. No PCA antibody was detected before week 6, then it appeared abruptly, remained elevated for lo-12 weeks and thereafter was no longer demonstrable. Reinfection on week 36 resulted in an abrupt secondary response in one animal, a delayed secondary response in another, and no response in the third. There was no correlation of PCA titers and flocculating antibody titers. Rabbit No. 1472, the animal which showed both a primary and an abrupt secondary response, was the weakest reactor in the slide flocculation test with all three antigens. The results obtained with the 4 rabbits
infected with 5000 cercariae are shown in Fig. 4. Three developed PCA antibody beginning at week 6 and reached a peak titer by week 9-13. Two of the three animals showed a secondary response equal to, or greater than, the original PCA titer following reinfection at week 36. The anaphylactic antibody response of the rabbits infected with 25,000 cercariae is depicted in Fig. 5. Three of the four animals had demonstrable PCA activity; in two the primary response was biphasic, with an initial short-lived appearance of antibody about week 6, followed by a more prolonged elevation from weeks 12-19. Following reinfection in all three animals which showed a primary response, there was a prompt reappearance of anaphylactic antibody. The one rabbit which did not show a primary response did not demonstrate PCA activity after reinfection. The patterns of anaphylactic antibodies in the three groups of animals are compared
ZVAIFLER,
282
SADUN, BECKER, 1000
500
AND SCHOENBECHLER CERCARIAE
400 300
45.j """""". ,472 ..-... 1477-
200
PCA TITER
loo 90 80
70 60 50 40
WEEKS
FIG. 3. The individual titers of homologous passive cutaneous anaphylactic antibody obtained 3 rabbits following infection with 1000 cercariae of S. mansoni. Reinfection with 5000 cercariae week 36 is designated by the arrow.
5000
in on
CERCARIAE ,606 -..-..,679
,Y"""."""""
1696 ,699
."""""""".
PCA TITER
0
4
8
12
16
20
24
26
32
36
40
44
46
52
56
60
WEEKS FIG. 4. The individual titers of homologous passive cutaneous anaphylactic antibody obtained 4 rabbits following infection with 5000 cercariae of S. mansoni. Reinfection with 5000 cercariae week 36 is designated by the arrow.
in on
ANTIBODY
DEMONSTRATION 25,000
IN INFECTED
RABBITS
283
CERCARIAE
1700 .-..I ,701 ~~~~~~~. 1702 --170 PCA TITER
0
4
6
I2
16
20
24
,26
32
36
40
44
46
62
56
60
WEEKS
FIG. 5. The individual titers of homologous passive cutaneous anaphylactic rabbits following infection with 25,000 cercariae of s. munsoni. Reinfection week 36 is designated by the arrow.
in Fig. 6. Antibody activity was seldom present before week 6, following the initial infection. In general, the group infected with 1000 cercariae showed an abrupt rise to the highest PCA titers which then fell rapidly. The animals infected with 25,000 cercariae had the lowest titers, which remained elevated for the longest period of time. The response of the group infected with 5000 cercariae was intermediate. The PCA titers in all three groups declined by week 20. With reinfection there was a more abrupt appearance of antibody, which in most cases remained elevated throughout the next 20 weeks. The secondary responses of the rabbits infected with 5000 and 25,000 cercariae were equal to, or greater than, the initial response. In the group infected with 1000 cercariae, the secondary response was less evident than the primary one. Nine of the eleven infected rabbits demonstrated anaphylactic antibody. The two animals which did not show a primary
antibody obtained in 4 with 5000 cercariae on
response did not develop anaphylactic antibody following reinfection. Two additional animals which had anaphylactic activity after the initial infection did not show a secondary response after reinfection. There was no correlation of the two anaphylactic antibody titers with the flocculating antibody titers. Susceptibility of Flocculating and Anaphylactic Antibody to Heat and Mercaptoethanol Treatment
Heating serum at 56°C for 60-120 minutes resulted in a reduction of anaphylactic acitivty by 50-75 % . Anaphylactic activity was completely destroyed by heating beyond 2 hours. Similar treatment had no effect on flocculating antibody titers. Exposure of serum to 0.1 M 2-mercaptoethanol followed by alkylation with an excess of 0.02 M iodoacetamide resulted in complete loss of anaphylactic activity, but there was no effect on flocculating: antibodv.s _._ _.---
284
ZVAIFLER,
xo4mm-
SADUN,
BECKER,
25.000
AND
SCHOENBECHLER
CERCARIAE
ITO2 _1_ 110 -
mPCA TITFR
WEEKS
FIG. 6. A composite showing the patterns of development of homologous anaphylactic antibody in rabbits infected with 1000, 5000 and 25,000 cercariae of S. mansani, and when reinfected with 5000 cercariae on week 36.
ANTIBODY
DEMONSTRATION
DISCUSSION
The results of these studies indicate that there are many features distinguishing the flocculating from the anaphylactic antibodies which develop in rabbits with schistosomiasis. All of the animals produced flocculating antibodies which appeared by weeks 3-4, and persisted throughout the study. Anaphylactic antibody was present by weeks 6-8 in nine of eleven infected rabbits; it persisted for several weeks in some, but had declined by week 20 in all. Although there was a direct correlation between the number of infecting organisms and the height of flocculating antibody titers, no such correlation was noted for anaphylactic antibody. When the rabbits were reinfected on week 36, there was very little increase in flocculating antibody titers, but seven of eleven rabbits showed a secondary anaphylactic antibody response. Flocculating antibodies are heat stable, not sensitive to mercaptoethanol and appear in the 7s gamma G globulin fraction on DEAE cellulose chromatography. Anaphylactic antibody is destroyed by heating, is inactivated by reduction and alkylation (Zvaifler and Becker, 1966), and is eluted from DEAE cellulose after the flocculating antibody (Zvaifler, unpublished abstract). Our results with relation to the time course of development of anaphylactic antibody in rabbits with schistosomiasis were remarkably similar to those found with rats (Ogilvie et al., 1966). Reaginic-type antibodies were found in the serum of rats 4 weeks after infection with S. mansoni. These persisted until week 12, and then disappeared. Sera taken one week after a second exposure were strongly positive. Monkeys showed reagins 6-16 weeks after exposure, and became negative within 4 weeks. All sera taken one week after reexposure were positive, and in some remained so for up to 20 weeks. Infection
IN
INFECTED
RABBITS
285
with irradiated cercariae resulted in negative or only weakly positive results even with repeated exposure (Ogilvie et al., 1966). Hsu and Hsu (1966) reported somewhat similar results in monkeys infected with S. japonicum. They found that 5 of 15 monkeys developed PCA positive sera; 2 in 38 days, 2 in 188 days and 1 in 592 days after the first inoculation. In contrast to Ogilvie’s findings they identified reagin-like antibody in the sera of 23 of 38 monkeys exposed to X-irradiated cercariae of S. japonicum. The antibodies did not appear in any of the animals before day 60 of infection, and in most they were not seen until many months or years, and only multiple reinfections. The situation in man and chimpanzees has so far been studied only in a limited number of specimens. Serum from individuals infected with S. mansoni or S. haematobium gave PCA reactions in the skin of rhesus monkeys (Sadun et al., 1966). The persistence of anaphylactic antibody in helminthic infections and the secondary response after reinfection differs from that which is seen when rabbits are immunized with soluble protein and haptene-protein antigens (Zvaifler and Becker, 1966). A primary immunization with soluble protein antigens in Freund’s complete or incomplete adjuvant is followed in 6-10 days by the appearance of low titers of anaphylactic antibody in one-half to two-thirds of the animals. The antibody rapidly disappears and is not recalled by subsequent immunization. A very similar situation has been reported in rats (Mota, 1964). The reason for the greater percentage of animals developing anaphylactic antibody, the higher PCA titers and the persistence of the antibody in animals with schistosomiasis is not known. The living antigen with its complex life cycle may result in a prolonged period of antigenic stimulation. The long latent period between exposure and the ap-
286
ZVAIFLER,
SADUN,
BECKER,
pearance of anaphylactic antibody may mean that the parasite does not become antigenic until it reaches adulthood, or that the antigen has arrived at some anatomic locus, such as the mesentery, favorable for anaphylactic antibody production. It is of interest that reaginic type antibodies are usually associated with diseases characterized by an eosinophilic response in the blood, i.e., allergy and helminthic infections, and eosinophilia becomes prominent in the organs of rabbits 30-40 days after infection by schistosomes (Koppisch, 1937). This is approximately the time when anaphylactic antibody appears in the serum. Recent studies have shown that passive cutaneous anaphylaxis is mediated by two different kinds of antibodies. In the first, serum from one species produces PCA reactions in the skin of another species, e.g., rabbit serum in guinea pig skin. This type of reaction, heterologous anaphylaxis or heterocytotropic anaphylaxis, is mediated by 7s gamma G globulin. This class of antibody is not affected by heating, is only mildly inactivated by reduction and alkylation, and is isolated in the “fall through” peak on DEAE-cellulose chromatograph (Zvaifler and Barbaro, 1967). Passive cutaneous antibody reactions are obtained within hours after the introduction of the antisera into the skin of the heterologous species, but are negative after a latent period of 48 hours, presumably because the antibody is unable to remain fixed to the skin sites. This is the type of anaphylactic antibody described by Ivey and Slanga (1965) who showed that serum from rabbits infected with Trichinella or Toxocara produced PCA reactions in guinea pig skin. The flocculating antibodies in schistosomiasis belong to this class of antibodies. The second kind of anaphylactic antibody gives passive cutaneous anaphylactic reactions in the skin of the species from which the antibody is derived, i.e.,
AND
SCHOENBECHLER
rabbit antibody in rabbit skin. This reaction is called homologous anaphylaxis or homocytotropic anaphylaxis, and is mediated by antibodies which differ from the conventional 7s immunoglobulins. Rabbit homologous anaphylactic antibody is heat labile, inactivated by reduction and alkylation, migrates as a beta globulin, and is eluted from DEAB-cellulose after the gamma G globulins. When placed in rabbit skin there is a latent period of 48-72 hours before PCA reactions occur, but the antibody remains fixed to the skin for many days or weeks (Zvaifler and Becker, 1966). Studies are in progress to determine whether the homologous anaphylactic antibody described in this report plays any role in the immunity which develops in rabbits infected with Schistosoma mansoni. ACKNOWLEDGMENTS The technical help of Mrs. Jennifer is gratefully acknowledged.
Robinson
REFERENCES ANDERSON, R. Schistosoma
I. 1960. Serologic diagnosis of mansoni infections. I. Development of a cercarial antigen slide flocculation test. American Journal of Tropical Medicine and Hygiene
9, 299-303.
Hsu, H. F., AND Hsu, S. Y. Lr 1966. Reagin-like antibody in rhesus monkeys immune to Schistosoma japonicum. Zeitschrift fur Tropenmedizin und Parasitologic 17, 166-176. IVEY, M. H., AND SLANGA, R. 1965. An evaluation
of passive cutaneous anaphylaxis reactions with Trichinella and Toxocara antibodyantigen systems. American Journal of Tropical Medicine and Hygiene 14, 1052-1056. KAGAN, I. G., AND PELLEGRINO, J. 1961. A critical review of immunological methods for the diagnosis of bilharziasis. Bulletin World Health Organization 25, 61 l-674. KOPPISCH, E. 1937. Studies on schistosomiasis mansoni in Puerto Rico. II. The pathological anatomy of experimental schistosomiasis mansoni in the rabbit and albino rat. Puerto
ANTIBODY Rico Journal of Public Medicine 13, l-59.
DEMONSTRATION
Health
and
Tropical
I. 1964. The mechanism of anaphylaxis. I. Production and biologic properties of mast cell sensitizing antibody. Immunology 7, 681699. OGILVIE, B. M. 1964. Reagin-like antibodies in animals immune to helminth parasites. Nature MOTA,
204, 91-92. OGILVIE,
B. M.,
SMITHERS,
S. R., AND TERRY,
R. J.
1966. Reagin-like antibodies in experimental infections of Schistosoma mansoni and the passive transfer of resistance. Nature 209, 1221-1223. SADUN,
E. H., SCHOENBECHLER,
M. 1965. Multiple
M.
J., AND BENTZ, response in Schis-
antibody infections: Antigenic constituents in eggs, cercariae and adult (excretions and secretions) determined by flocculation reactions, cross absorption and double tosoma
mansoni
IN
INFECTED
RABBITS
287
diffusion studies. American Journal of Tropical Medicine and Hygiene 14, 977-995. SADUN, E. H., VON LICHTENBERG, F., HICKMAN, R. L., BRUCE, 3. I., SMITH, I. H., AND SCHOENBECHLER, M. J. 1966. Schistosomiasis mansoni in the chimpanzee: Parasitologic, clinical, serologic, pathologic and radiologic observations. American Journal of Tropical and Hygiene 15, 496-506. J., AND BECKER, E. L. 1966. Rabbit anaphylactic antibody. Journal of Experimental Medicine 123, 935-950. ZVAIFLER, N. J., SADUN, E. H., AND BECKER, E. L. Medicine ZVAIFLER, N.
1966. Anaphylactic (reaginic) antibodies in helminthic infections. Clinical Research 45, 336 (abstr.). ZVAIFLER, N. I., AND BARBARO, J. 1967. Anaphylactic antibody in the primary response of rabbits. International Archives of Allergy and Applied
Immunology,
31, 465474.