EXPEHlhlENTAL PARASITOLOGY 41, 160-166
Schistosoma
ALAN
(1977)
mansoni: lmmunoglobulins Involved Immunization of Laboratory Mice SMITHERS, PAMELA AND KENNETH BROOMFIELD
SHER,~ S. RONALD
in Passive
MACKENZIE,
Division of Parasitobgy, National Institute for Medical Research, Mill Hill, London NW7 IAA, England, U.K. (Accepted
for publication
12 April
1976)
SHER, ALAN, SMITHERS, S. RONALD, MACKENZIE, PAMELA, AND BROOMFIELD, KENNETH. 1977. Schistosoma numsoni: Immunoglobulins involved in passive immunization of laboratory mice. Experimental Parasitology 41, 160-166. Sera obtained from mice 12-16 weeks after a single infection of Schistosoma mansoni were separated by gel filtration or DEAEcellulose chromatography and the fractions were tested for their activity in passively immunizing normal recipients. Partial protection was found associated with the 7s fraction obtained by gel filtration on Sephadex G-200. No activity was detectable in either the 19s or 8s regions of the column profile, When sera were separated by ammonium sulfate precipitation and DEAE-cellulose chromatography, fractions containing large amounts of IgGl with respect to IgGz gave the best passive protection. In contrast, an antibody activity known to passively confer a delay in the migration of schistosomula in the absence of significant loss of parasite viability was found to associate with a fraction enriched in IgGa immunoglobulin. INDEX DESCRIPTORS: Schistosomu mansoni; trematode; blood fluke; Mice; Acquired immunity; Passive immunization; Immunoglobulins; IgG.
INTRODUCTION
Laboratory mice infected with Schistosoma munsoni acquire a partial resistance to subsequent challenge infection (Olivier and Schneidermann 1953; Sher et al. 1974). The immunologic mechanism responsible for the destruction of challenge infections in the resistant mouse appears to involve the cooperation of several humoral and cellular components. In a previous study, we identified one of these, a humoral component, by demonstrating that sera from immune (12- to 16-week infected) mice could passively immunize normal recipients against subsequent schistosome challenges 1 Present address: Department of Pathology, Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, Mass. 02115, U.S.A.
(Sher et al. 1975). The transfer of protection was only partial with respect to the level of immunity displayed by the donors and required the injection of large volumes of immune sera. The serum activity responsible for passive immunization appeared to be directed primarily against the early larval stages of the challenge infection, We based this hypothesis on the finding that immune serum brought about a reduction in the recovery of schistosomula from the lungs 4-5 days after challenge as well as a subsequent decrease in final adult worm burden. Moreover, the effectiveness of immune serum was considerably reduced when administered to recipients several days after, as opposed to shortly before the challenge infection.
160 Copyright All rights
c 1977 by Academic oP reproduction in any
Press, Inc. form reserved.
ISSN
0014
4894
l(il
Schistosoma mansoni: PASSlVE I~I,\fUSIZA'l-ION
In addition to protective immunity, a second phenomenon influencing the fate of challenge infections in the immunized mouse was found to involve a humoral mechanism. This phenomenon, a delay in the migration of schistosomula to the lungs induced during the first 3 weeks of the primary infection (Sher et al. 1974) could also be produced in normal recipients by transferring serum from the appropriate immunized donors (Sher et al. 1975). In the present study, we have attempted to identify and compare the factors in the sera of schistosome-infected mice responsible for passive transfer of protective immunity and delayed migration of schistosomules. The results of these experiments suggest that IgG antibodies mediate both phenomena. However, these antibodies appear to belong to distinguishable immunoglobulin subclasses,a finding which may explain the differing biological activities which they possess.
described previously (Sher et al. 1975). Donor animals were given percutaneous infections of 3045 cercariae each and bled 12-15 weeks later. Of the nine serum pools collected, five were found to possess significant protective activity. In another experiment, serum was obtained from mice also given 3045 cercariae each but bled 3 weeks after infection. The origins of the six active serum pools used in the study arc listed in Table I. Fraction&ions
Sephadex G-200. Samples (6-10 ml) of mouse sera were eluted from a column (125 x 3.5 cm) of Sephadex G-200 (Pharmacia) equilibrated in phosphate-buffered saline, pH 7.4 (PBS ). DEAE-cellulose. Serum pools (6-10 ml) were diluted one to one in PBS and an equal volume of saturated ammonium sulfate was added slowly with stirring. The precipitates which formed after standing for 2 hr at 4 C were then collected by cenMATERJALSANDMETHODS trifugation and washed twice in 20 vol of Parasite and Experimental Hosts one-half saturated ammonium sulfate. The The Puerto Rican strain of Schistosoma pellet fraction was dissolved in 0.005 M jnansoni was used in all experiments Tris-phosphate buffer, pH 8.6, and dialyzed (Smithers and Terry 1965). Female inbred against several changes of the same buffer. CBA and C3H mice and outbred Parkes The conditions for DEAE-cellulose chromamice were obtained from our Institute tography are those described by Potter ( 1967). Ammonium sulfate-purified serum stocks. fractions were applied to columns (30 x Serum Preparation 2.5 cm) of DE-52 (Whatman) equilibrated Serum pools from mice with acquired im- in 0.005 M Tris-phosphate buffer, pH 8.6. munity to S. mansoni were prepared as Protein was eluted stepwise with 0.0275 M TABLE
I
Serum Pools from Mice Infected with Schisloso?namansoni Used in Fractionation Experiments Serum pool
Donor mouse strain
Period of infection (weeks)
Final volume of each fraction _Volume of starting serum sample
Recipient mouse strain
A B C D E F
Parkes CBA CBA Parkes Parkes Parkes
12-13 13 12-13 12-14 13 3
1.0 2.0 1.4 1.0 0.9 1.8
Parkes C3H CBA CBA CBA Parkes
162
SHER ET AL.
Tris-phosphate, pH 7.7, followed by 2 AI NaCl in 0.0005 M Tris-phosphate, pH 8.6. All fractionation steps were performed at 4 C. The optical density of eluates was measured at 280 nm in a Uvicord (LKB Instruments) recording spectrophotometer. Column fractions were pooled, dialyzed against PBS when necessary, and concentrated by surrounding the dialysis sacs with Aquacide II ( Calbiochem). The final volume of each fraction with respect to the volume of the starting serum sample is presented in Table I. Estimation
of Mouse lmmunoglohulins
Rabbit antisera specific for mouse IgM, IgA, I&1, and IgG (IgG and W&t,) immunoglobulins were kindly provided by Drs. D. W. Dresser and R. M. E. Parkhouse. These reagents had been prepared by immunization with purified myeloma proteins or their Fc fragments and had been absorbed with immunoglobulins of other classes to remove contaminating cross-reactions. Mouse IgA and IgM were detected in serum fractions by double diffusion in 1% agar (Ouchterlony 1958). Levels of mouse IgGl and IgG2 in sera and their chromatographic fractions were estimated by single radial irnmunodiffusion (Man&i et al. 1965) in 1.5% agar. The concentration of immunoglobulin of each subclass was calculated from a standard curve obtained by analyzing the reaction of different concentrations of purified myeloma protein with its respective monospeciflc antiserum. The purity of chromatographic fractions was analyzed by means of immunoelectrophoresis in 1% agar (Grabar and Williams 1953). The developing reagent ( a gift of Dr. D. W. Dresser) was an antiserum prepared by immunizing rabbits with unfractionated normal mouse serum. Tests for Protection and Delayed Migration Infections
of Challenge
The protective activity of immune serum fractions was determined by assaying their
capacity to passively immunize normal mice against challenge infections of S. mansoni (Sher et al. 1975). Mice in groups of 10 were each injected intravenously in the tail vein with 0.5 ml of the test sample. Control animals received the same volume of normal mouse serum or an equivalent chromatographic fraction thereof. On the following day, the mice were exposed percutaneously to 125-140 cercariae each. The recovery of the challenge infection was determined 6-7 weeks later by assaying the number of adult worms obtained by perfusion of the hepatic portal system (Smithers and Terry 1965). The presence of an activity which delays the migration of schistosomula to the lungs was assayed in fractions of serum obtained from 3-week infected mice as follows: Mice in groups of five or six animals were each injected intravenously with 0.5 ml of serum or serum fraction and on the following day challenged percutaneously with 500 cercariae. On the fourth day after challenge the animals were sacrificed and the recovery of schistosomula from the lungs was assayed as described previously (Sher et al. 1974). The control animals in this experiment were mice which had been injected with normal mouse serum.
RESULTS Transfer of Protection with Immune Serum Fractions Obtained by Sephadex Gel Filtration Two different pools (A and B) of immune mouse serum were separated by gel filtration on a Sephadex G-200 column. The eluate from Pool A was divided into three fractions (A-l, A-2, A-3) representing each of the three major protein peaks recorded on the optical density profile (Fig. 1A). Recipients of each fraction were challenged and the recovery of the challenged infection was assayed 6 weeks later by liver perfusion. The results (Table II, indicated that only one of the fractions tested, fraction A-2, transferred significant resistance
Schistosoma manson!: PASSIVE IMMUNIZATION to challenge. This fraction is known to contain a mixture of 7s IgG, 7s IgA, and 8s IgE immunoglobulins. IgM was not detected in this fraction by our antiserum although it is possible that the monomeric form of the immunoglobulin may have been present in trace quantity. A second gel filtration experiment was designed to test the protective activities of fractions known to be selectively enriched in either 7s IgG or 8s IgE. In that experiment employing Pool B, the second protein peak eluted from the G-200 column was subdivided into two fractions, the first the IgE molecular comprising (B-2a) weight region and the second (B-2b) consisting of the portion of the peak containing the bulk of the 7s immunoglobulin (Fig. 1). Significant passive protection was again obtained with only one of the fractions tested (Table II). This was the fraction (B-2b) known to be enriched in 7s immunoglobulins. Protective Activities of Serum Fractions Obtained by Ammonium Sulfate Precipitation and Ion-Exchange Chromatography In order to identify further the factor responsible for passive protection, three serum pools (C, D, and E) were separated by precipitation in ammonium sulfate, followed by DEAE-cellulose chromatography. This procedure yielded three fractions. The first, the supernatant from the ammonium sulfate precipitation step is known to consist largely of nonimmunoglobulin proteins (Potter 1967). The second and third fractions (C, D, E-l; C, D, E-2) were the two protein peaks obtained by separating the ammonium sulfate pellet on DEAE-cellulose (Fig. 1B). When analyzed by immunoelectrophoresis against a rabbit antiserum to whole mouse serum, the first of the DEAE fractions consistently gave a single precipitin arc co-migrating with purified IgG mouse myeloma protein. The second DEAE fraction, when assayed by the same procedure was found to contain IgG, IgA,
163 A GZOO
Fr
1
Fr. 2 I , , 2a , 2b
ij P4 1’
I,
F,
3
/,’,/:I’ ‘\ .- --!Lh-VOLUME
Fr 1
F, 2
I3 DEAE
VOLUME
FIG. 1. RepresentativeSephadexG-200 (A) and DEAE-cellulose (B) column elution profiles of sera from mice infected with Schistosomamansoni. The horizontal bars denote the regions of the profile pooled to form each fraction. The arrows indicate the points of application of chromatographic buffers. and IgM as well as several other nonimmunoglobulin proteins. Analysis by radial immunodiffusion revealed that unseparated immune serum as well as both of the DEAE fractions contained levels of IgGl immunoglobulin which were significantly elevated with respect to the levels present in normal (uninfected) mouse serum (Table III), In contrast, only a small increase in the levels of IgGz immunoglobulin could be detected. The first of the DEAE fractions was found to contain the bulk (72-74s ) of the IgG, eluted from the columns. In the single instance where the ammonium sulfate supematant was tested (Pool C), no protective activity was detected in that fraction (Table III). Instead, significant passive immunity was consistently found in mice given the second of
164
SHER ET AL. TABLE Protective
Activities
Fraction
Al A2 A3 Normal
II
of Sephadex G-200 Fractions of Serum Pools from Mice Infected with Schistosoma mansoni Adult worm recovery (mean f SD)”
serum
Bl B2a B2b B3 2b of normal serum
+ -I-
NTd
NT
8.3 5.4 4.9 7.5
NSC
Schistosoma
ALAN
(1977)
mansoni: lmmunoglobulins Involved Immunization of Laboratory Mice SMITHERS, PAMELA AND KENNETH BROOMFIELD
SHER,~ S. RONALD
in Passive
MACKENZIE,
Division of Parasitobgy, National Institute for Medical Research, Mill Hill, London NW7 IAA, England, U.K. (Accepted
for publication
12 April
1976)
SHER, ALAN, SMITHERS, S. RONALD, MACKENZIE, PAMELA, AND BROOMFIELD, KENNETH. 1977. Schistosoma numsoni: Immunoglobulins involved in passive immunization of laboratory mice. Experimental Parasitology 41, 160-166. Sera obtained from mice 12-16 weeks after a single infection of Schistosoma mansoni were separated by gel filtration or DEAEcellulose chromatography and the fractions were tested for their activity in passively immunizing normal recipients. Partial protection was found associated with the 7s fraction obtained by gel filtration on Sephadex G-200. No activity was detectable in either the 19s or 8s regions of the column profile, When sera were separated by ammonium sulfate precipitation and DEAE-cellulose chromatography, fractions containing large amounts of IgGl with respect to IgGz gave the best passive protection. In contrast, an antibody activity known to passively confer a delay in the migration of schistosomula in the absence of significant loss of parasite viability was found to associate with a fraction enriched in IgGa immunoglobulin. INDEX DESCRIPTORS: Schistosomu mansoni; trematode; blood fluke; Mice; Acquired immunity; Passive immunization; Immunoglobulins; IgG.
INTRODUCTION
Laboratory mice infected with Schistosoma munsoni acquire a partial resistance to subsequent challenge infection (Olivier and Schneidermann 1953; Sher et al. 1974). The immunologic mechanism responsible for the destruction of challenge infections in the resistant mouse appears to involve the cooperation of several humoral and cellular components. In a previous study, we identified one of these, a humoral component, by demonstrating that sera from immune (12- to 16-week infected) mice could passively immunize normal recipients against subsequent schistosome challenges 1 Present address: Department of Pathology, Peter Bent Brigham Hospital, 721 Huntington Avenue, Boston, Mass. 02115, U.S.A.
(Sher et al. 1975). The transfer of protection was only partial with respect to the level of immunity displayed by the donors and required the injection of large volumes of immune sera. The serum activity responsible for passive immunization appeared to be directed primarily against the early larval stages of the challenge infection, We based this hypothesis on the finding that immune serum brought about a reduction in the recovery of schistosomula from the lungs 4-5 days after challenge as well as a subsequent decrease in final adult worm burden. Moreover, the effectiveness of immune serum was considerably reduced when administered to recipients several days after, as opposed to shortly before the challenge infection.
160 Copyright All rights
c 1977 by Academic oP reproduction in any
Press, Inc. form reserved.
ISSN
0014
4894
l(il
Schistosoma mansoni: PASSlVE I~I,\fUSIZA'l-ION
In addition to protective immunity, a second phenomenon influencing the fate of challenge infections in the immunized mouse was found to involve a humoral mechanism. This phenomenon, a delay in the migration of schistosomula to the lungs induced during the first 3 weeks of the primary infection (Sher et al. 1974) could also be produced in normal recipients by transferring serum from the appropriate immunized donors (Sher et al. 1975). In the present study, we have attempted to identify and compare the factors in the sera of schistosome-infected mice responsible for passive transfer of protective immunity and delayed migration of schistosomules. The results of these experiments suggest that IgG antibodies mediate both phenomena. However, these antibodies appear to belong to distinguishable immunoglobulin subclasses,a finding which may explain the differing biological activities which they possess.
described previously (Sher et al. 1975). Donor animals were given percutaneous infections of 3045 cercariae each and bled 12-15 weeks later. Of the nine serum pools collected, five were found to possess significant protective activity. In another experiment, serum was obtained from mice also given 3045 cercariae each but bled 3 weeks after infection. The origins of the six active serum pools used in the study arc listed in Table I. Fraction&ions
Sephadex G-200. Samples (6-10 ml) of mouse sera were eluted from a column (125 x 3.5 cm) of Sephadex G-200 (Pharmacia) equilibrated in phosphate-buffered saline, pH 7.4 (PBS ). DEAE-cellulose. Serum pools (6-10 ml) were diluted one to one in PBS and an equal volume of saturated ammonium sulfate was added slowly with stirring. The precipitates which formed after standing for 2 hr at 4 C were then collected by cenMATERJALSANDMETHODS trifugation and washed twice in 20 vol of Parasite and Experimental Hosts one-half saturated ammonium sulfate. The The Puerto Rican strain of Schistosoma pellet fraction was dissolved in 0.005 M jnansoni was used in all experiments Tris-phosphate buffer, pH 8.6, and dialyzed (Smithers and Terry 1965). Female inbred against several changes of the same buffer. CBA and C3H mice and outbred Parkes The conditions for DEAE-cellulose chromamice were obtained from our Institute tography are those described by Potter ( 1967). Ammonium sulfate-purified serum stocks. fractions were applied to columns (30 x Serum Preparation 2.5 cm) of DE-52 (Whatman) equilibrated Serum pools from mice with acquired im- in 0.005 M Tris-phosphate buffer, pH 8.6. munity to S. mansoni were prepared as Protein was eluted stepwise with 0.0275 M TABLE
I
Serum Pools from Mice Infected with Schisloso?namansoni Used in Fractionation Experiments Serum pool
Donor mouse strain
Period of infection (weeks)
Final volume of each fraction _Volume of starting serum sample
Recipient mouse strain
A B C D E F
Parkes CBA CBA Parkes Parkes Parkes
12-13 13 12-13 12-14 13 3
1.0 2.0 1.4 1.0 0.9 1.8
Parkes C3H CBA CBA CBA Parkes
162
SHER ET AL.
Tris-phosphate, pH 7.7, followed by 2 AI NaCl in 0.0005 M Tris-phosphate, pH 8.6. All fractionation steps were performed at 4 C. The optical density of eluates was measured at 280 nm in a Uvicord (LKB Instruments) recording spectrophotometer. Column fractions were pooled, dialyzed against PBS when necessary, and concentrated by surrounding the dialysis sacs with Aquacide II ( Calbiochem). The final volume of each fraction with respect to the volume of the starting serum sample is presented in Table I. Estimation
of Mouse lmmunoglohulins
Rabbit antisera specific for mouse IgM, IgA, I&1, and IgG (IgG and W&t,) immunoglobulins were kindly provided by Drs. D. W. Dresser and R. M. E. Parkhouse. These reagents had been prepared by immunization with purified myeloma proteins or their Fc fragments and had been absorbed with immunoglobulins of other classes to remove contaminating cross-reactions. Mouse IgA and IgM were detected in serum fractions by double diffusion in 1% agar (Ouchterlony 1958). Levels of mouse IgGl and IgG2 in sera and their chromatographic fractions were estimated by single radial irnmunodiffusion (Man&i et al. 1965) in 1.5% agar. The concentration of immunoglobulin of each subclass was calculated from a standard curve obtained by analyzing the reaction of different concentrations of purified myeloma protein with its respective monospeciflc antiserum. The purity of chromatographic fractions was analyzed by means of immunoelectrophoresis in 1% agar (Grabar and Williams 1953). The developing reagent ( a gift of Dr. D. W. Dresser) was an antiserum prepared by immunizing rabbits with unfractionated normal mouse serum. Tests for Protection and Delayed Migration Infections
of Challenge
The protective activity of immune serum fractions was determined by assaying their
capacity to passively immunize normal mice against challenge infections of S. mansoni (Sher et al. 1975). Mice in groups of 10 were each injected intravenously in the tail vein with 0.5 ml of the test sample. Control animals received the same volume of normal mouse serum or an equivalent chromatographic fraction thereof. On the following day, the mice were exposed percutaneously to 125-140 cercariae each. The recovery of the challenge infection was determined 6-7 weeks later by assaying the number of adult worms obtained by perfusion of the hepatic portal system (Smithers and Terry 1965). The presence of an activity which delays the migration of schistosomula to the lungs was assayed in fractions of serum obtained from 3-week infected mice as follows: Mice in groups of five or six animals were each injected intravenously with 0.5 ml of serum or serum fraction and on the following day challenged percutaneously with 500 cercariae. On the fourth day after challenge the animals were sacrificed and the recovery of schistosomula from the lungs was assayed as described previously (Sher et al. 1974). The control animals in this experiment were mice which had been injected with normal mouse serum.
RESULTS Transfer of Protection with Immune Serum Fractions Obtained by Sephadex Gel Filtration Two different pools (A and B) of immune mouse serum were separated by gel filtration on a Sephadex G-200 column. The eluate from Pool A was divided into three fractions (A-l, A-2, A-3) representing each of the three major protein peaks recorded on the optical density profile (Fig. 1A). Recipients of each fraction were challenged and the recovery of the challenged infection was assayed 6 weeks later by liver perfusion. The results (Table II, indicated that only one of the fractions tested, fraction A-2, transferred significant resistance
Schistosoma manson!: PASSIVE IMMUNIZATION to challenge. This fraction is known to contain a mixture of 7s IgG, 7s IgA, and 8s IgE immunoglobulins. IgM was not detected in this fraction by our antiserum although it is possible that the monomeric form of the immunoglobulin may have been present in trace quantity. A second gel filtration experiment was designed to test the protective activities of fractions known to be selectively enriched in either 7s IgG or 8s IgE. In that experiment employing Pool B, the second protein peak eluted from the G-200 column was subdivided into two fractions, the first the IgE molecular comprising (B-2a) weight region and the second (B-2b) consisting of the portion of the peak containing the bulk of the 7s immunoglobulin (Fig. 1). Significant passive protection was again obtained with only one of the fractions tested (Table II). This was the fraction (B-2b) known to be enriched in 7s immunoglobulins. Protective Activities of Serum Fractions Obtained by Ammonium Sulfate Precipitation and Ion-Exchange Chromatography In order to identify further the factor responsible for passive protection, three serum pools (C, D, and E) were separated by precipitation in ammonium sulfate, followed by DEAE-cellulose chromatography. This procedure yielded three fractions. The first, the supernatant from the ammonium sulfate precipitation step is known to consist largely of nonimmunoglobulin proteins (Potter 1967). The second and third fractions (C, D, E-l; C, D, E-2) were the two protein peaks obtained by separating the ammonium sulfate pellet on DEAE-cellulose (Fig. 1B). When analyzed by immunoelectrophoresis against a rabbit antiserum to whole mouse serum, the first of the DEAE fractions consistently gave a single precipitin arc co-migrating with purified IgG mouse myeloma protein. The second DEAE fraction, when assayed by the same procedure was found to contain IgG, IgA,
163 A GZOO
Fr
1
Fr. 2 I , , 2a , 2b
ij P4 1’
I,
F,
3
/,’,/:I’ ‘\ .- --!Lh-VOLUME
Fr 1
F, 2
I3 DEAE
VOLUME
FIG. 1. RepresentativeSephadexG-200 (A) and DEAE-cellulose (B) column elution profiles of sera from mice infected with Schistosomamansoni. The horizontal bars denote the regions of the profile pooled to form each fraction. The arrows indicate the points of application of chromatographic buffers. and IgM as well as several other nonimmunoglobulin proteins. Analysis by radial immunodiffusion revealed that unseparated immune serum as well as both of the DEAE fractions contained levels of IgGl immunoglobulin which were significantly elevated with respect to the levels present in normal (uninfected) mouse serum (Table III), In contrast, only a small increase in the levels of IgGz immunoglobulin could be detected. The first of the DEAE fractions was found to contain the bulk (72-74s ) of the IgG, eluted from the columns. In the single instance where the ammonium sulfate supematant was tested (Pool C), no protective activity was detected in that fraction (Table III). Instead, significant passive immunity was consistently found in mice given the second of
164
SHER ET AL. TABLE Protective
Activities
Fraction
Al A2 A3 Normal
II
of Sephadex G-200 Fractions of Serum Pools from Mice Infected with Schistosoma mansoni Adult worm recovery (mean f SD)”
serum
Bl B2a B2b B3 2b of normal serum
+ -I-
NTd
NT
8.3 5.4 4.9 7.5
NSC
